Oligonucleotides and analogs labeled with energy transfer dyes

ABSTRACT

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R 21 Z 1 C(O)R 22 R 28  where R 21  is a C 1-5  alkyl attached to the donor dye, C(O) is a carbonyl group, Z 1  is either NH, sulfur or oxygen, R 22  is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R 28  includes a functional group which attaches the linker to the acceptor dye.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/788,836, filed Feb. 26, 2004, now U.S. Pat. No. 7,169,939 which is acontinuation of U.S. application Ser. No. 10/014,743, filed Oct. 29,2001, now U.S. Pat. No. 6,849,745, which is a continuation of U.S.application Ser. No. 09/272,097, filed Mar. 18, 1999, now U.S. Pat. No.6,335,440, which is a continuation of U.S. application Ser. No.09/046,203, filed Mar. 23, 1998, now U.S. Pat. No. 5,945,526, which is acontinuation of U.S. application Ser. No. 08/726,462, filed Oct. 4,1996, now U.S. Pat. No. 5,800,996, which is a continuation-in-part ofU.S. application Ser. No. 08/642,330 filed May 3, 1996, now U.S. Pat.No. 5,863,727 and U.S. application Ser. No. 08/672,196, filed Jun. 27,1996, now U.S. Pat. No. 5,847,162, all of which are incorporated hereinby reference.

2. FIELD OF THE INVENTION

The present invention relates to fluorescent dyes and, morespecifically, energy transfer fluorescent dyes and their use.

3. DESCRIPTION OF RELATED ART

A variety of fluorescent dyes have been developed for labeling anddetecting components in a sample. In general, fluorescent dyespreferably have a high quantum yield and a large extinction coefficientso that the dye may be used to detect small quantities of the componentbeing detected. Fluorescent dyes also preferably have a large Stokesshift (i.e., the difference between the wavelength at which the dye hasmaximum absorbance and the wavelength at which the dye has maximumemission) so that the fluorescent emission is readily distinguished fromthe light source used to excite the dye.

One class of fluorescent dyes which has been developed is energytransfer fluorescent dyes. In general, energy transfer fluorescent dyesinclude a donor fluorophore and an acceptor fluorophore. In these dyes,when the donor and acceptor fluorophores are positioned in proximitywith each other and with the proper orientation relative to each other,the energy emission from the donor fluorophore is absorbed by theacceptor fluorophore and causes the acceptor fluorophore to fluoresce.It is therefore important that the excited donor fluorophore be able toefficiently absorb the excitation energy of the donor fluorophore andefficiently transfer the energy to the acceptor fluorophore.

A variety of energy transfer fluorescent dyes have been described in theliterature. For example, U.S. Pat. No. 4,996,143 and WO 95/21266describe energy transfer fluorescent dyes where the donor and acceptorfluorophores are linked by an oligonucleotide chain. Lee et al., NucleicAcids Research 20(10):2471-2483 (1992) describes an energy transferfluorescent dye which includes 5-carboxy rhodamine linked to4′-aminomethyl-5-carboxy fluorescein by the 4′-aminomethyl substituenton fluorescein.

Several diagnostic and analytical assays have been developed whichinvolve the detection of multiple components in a sample usingfluorescent dyes, e.g. flow cytometry (Lanier et al., J. Immunol.132:151-156 (1984)); chromosome analysis (Gray et al., Chromosoma73:9-27 (1979)); and DNA sequencing. For these assays, it is desirableto simultaneously employ a set of two or more spectrally resolvablefluorescent dyes so that more than one target substance can be detectedin the sample at the same time. Simultaneous detection of multiplecomponents in a sample using multiple dyes reduces the time required toserially detect individual components in a sample. In the case ofmulti-loci DNA probe assays, the use of multiple spectrally resolvablefluorescent dyes reduces the number of reaction tubes that are needed,thereby simplifying the experimental protocols and facilitating themanufacturing of application-specific kits. In the case of automated DNAsequencing, the use of multiple spectrally resolvable fluorescent dyesallows for the analysis of all four bases in a single lane therebyincreasing throughput over single-color methods and eliminatinguncertainties associated with inter-lane electrophoretic mobilityvariations. Connell et al., Biotechniques 5:342-348 (1987); Prober etal., Science 238:336-341 (1987), Smith et al., Nature 321:674-679(1986); and Ansorge et al., Nucleic Acids Research 15:4593-4602 (1989).

There are several difficulties associated with obtaining a set offluorescent dyes for simultaneously detecting multiple target substancesin a sample, particularly for analyses requiring an electrophoreticseparation and treatment with enzymes, e.g., DNA sequencing. Forexample, each dye in the set must be spectrally resolvable from theother dyes. It is difficult to find a collection of dyes whose emissionspectra are spectrally resolved, since the typical emission bandhalf-width for organic fluorescent dyes is about 40-80 nanometers (nm)and the width of the available spectrum is limited by the excitationlight source. As used herein the term “spectral resolution” in referenceto a set of dyes means that the fluorescent emission bands of the dyesare sufficiently distinct, i.e., sufficiently non-overlapping, thatreagents to which the respective dyes are attached, e.g.polynucleotides, can be distinguished on the basis of the fluorescentsignal generated by the respective dyes using standard photodetectionsystems, e.g. employing a system of band pass filters andphotomultiplier tubes, charged-coupled devices and spectrographs, or thelike, as exemplified by the systems described in U.S. Pat. Nos.4,230,558, 4,811,218, or in Wheeless et al., pgs. 21-76, in FlowCytometry: Instrumentation and Data Analysis (Academic Press, New York,1985).

The fluorescent signal of each of the dyes must also be sufficientlystrong so that each component can be detected with sufficientsensitivity. For example, in the case of DNA sequencing, increasedsample loading can not compensate for low fluorescence efficiencies,Pringle et al., DNA Core Facilities Newsletter, 1:15-21 (1988). Thefluorescent signal generated by a dye is generally greatest when the dyeis excited at its absorbance maximum. It is therefore preferred thateach dye be excited at about its absorbance maximum.

A further difficulty associated with the use of a set of dyes is thatthe dyes generally do not have the same absorbance maximum. When a setof dyes are used which do not have the same absorbance maximum, a tradeoff is created between the higher cost associated with providingmultiple light sources to excite each dye at its absorbance maximum, andthe lower sensitivity arising from each dye not being excited at itsabsorbance maximum.

In addition to the above difficulties, the charge, molecular size, andconformation of the dyes must not adversely affect the electrophoreticmobilities of the fragments. The fluorescent dyes must also becompatible with the chemistry used to create or manipulate thefragments, e.g., DNA synthesis solvents and reagents, buffers,polymerase enzymes, ligase enzymes, and the like.

Because of the multiple constraints on developing a set of dyes formulticolor applications, particularly in the area of four color DNAsequencing, only a few sets of fluorescent dyes have been developed.Connell et al., Biotechniques 5:342-348 (1987); Prober et al., Science238:336-341 (1987); and Smith et al., Nature 321:674-679 (1986).

One class of fluorescent dyes that has been found to be useful inmulticolor applications are rhodamine dyes, e.g., tetramethylrhodamine(TAMRA), rhodamine X (ROX), rhodamine 6G (R6G), rhodamine 110 (R110),and the like. U.S. Pat. No. 5,366,860. Rhodamine dyes are particularlyattractive relative to fluorescein dyes because (1) rhodamines aretypically more photostable than fluoresceins, (2) rhodamine-labeleddideoxynucleotides are better substrates for thermostable polymeraseenzymes, and (3) the emission spectra of rhodamine dyes is significantlyto the red (higher wavelength) of fluoresceins.

One drawback associated with currently available rhodamine dyes,particularly in the context of multiplex detection methods, is therelatively broad emission spectrum of the rhodamine dyes. This broademission spectrum limits spectral resolution between spectrallyneighboring dyes, making the multicomponent analysis of such dyecombinations difficult. A second drawback associated with currentlyavailable rhodamine dyes is that their absorption spectrum does notmatch the wavelength of currently available solid statefrequency-doubled green diode lasers, e.g., neodymium solid-state YAGlasers, which have an emission line at approximately 532 nm. It ishighly advantageous to use such lasers because of their compact size,long useful life, and efficient use of power.

Energy transfer fluorescent dyes possess several features which makethem attractive for use in the simultaneous detection of multiple targetsubstances in a sample, such as in DNA sequencing. For example, a singledonor fluorophore can be used in a set of energy transfer fluorescentdyes so that each dye has strong absorption at a common wavelength.Then, by varying the acceptor fluorophore in the energy transfer dye, aseries of energy transfer dyes having spectrally resolvable fluorescenceemissions can be generated.

Energy transfer fluorescent dyes also provide a larger effective Stokesshift than non-energy transfer fluorescent dyes. This is because theStokes shift for an energy transfer fluorescent dye is based on thedifference between the wavelength at which the donor fluorophoremaximally absorbs light and the wavelength at which the acceptorfluorophore maximally emits light. In general, a need exists forfluorescent dyes having larger Stokes shifts.

The sensitivity of any assay using a fluorescent dye is dependent on thestrength of the fluorescent signal generated by the fluorescent dye. Aneed therefore exists for fluorescent dyes which have a strongfluorescence signal. With regard to energy transfer fluorescent dyes,the fluorescence signal strength of these dyes is dependent on howefficiently the acceptor fluorophore absorbs the energy emission of thedonor fluorophore. This, in turn, depends on a variety of variables,including the proximity of the donor fluorophore to the acceptorfluorophore and the orientation of the donor fluorophore relative to theacceptor fluorophore. A need therefore exists for energy transferfluorescent dyes in which the orientation between the donor and acceptorfluorophore is such that energy is efficiently transferred between thedonor and acceptor fluorophore.

4. SUMMARY OF THE INVENTION

The present invention relates to linkers for linking a donor dye to anacceptor dye in an energy transfer fluorescent dye. The presentinvention also relates to energy transfer fluorescent dyes havingenhanced fluorescence. The present invention also relates to reagentswhich include the energy transfer dyes of the present invention, methodsfor using the dyes and reagents, and kits within which the dyes andreagents are included.

One linker according to the present invention for linking a donor dye toan acceptor dye in an energy transfer fluorescent dye has the generalstructure R₂₁Z₁C(O)R₂₂R₂₈, as illustrated below, where R₂₁ is a C₁₋₅alkyl attached to the donor dye, C(O) is a carbonyl group, Z₁, is eitherNH, sulfur or oxygen, R₂₂ is a substituent attached to the carbonylcarbon which may be either an alkene, diene, alkyne, a five or sixmembered ring having at least one unsaturated bond or a fused ringstructure, and R₂₈ includes a functional group which attaches the linkerto the acceptor dye.

The R₂₈ group used in the linker may be any group known in the art whichcan be used to attach the R₂₂ group to an acceptor dye. Typically, theR₂₈ group will be attached to a benzene ring or other aromatic ringstructure on the acceptor dye. Accordingly, R₂₈ is preferably formed byforming an electrophilic functional group on the benzene ring or otheraromatic ring structure of the acceptor dye, such as a carboxylic acids,acid halide, sulfonic acid, ester, aldehyde, thio, disulfide,isothiocyanate, isocyanate, sulfonyl halide, maleimide,hydroxysuccinimide ester, haloacetyl, hydroxysulfosuccinimide ester,imido ester, hydrazine, azidonitrophenyl, and azide. The R₂₂ group canthen be added to the acceptor dye, either before or after attachment ofthe donor dye to the R₂₂ group, by reacting the electrophilic agent onthe acceptor dye with a nucleophile, such as an amino, hydroxyl orsulfhydryl nucleophile.

For example, in the embodiment illustrated below, the linker has thegeneral structure R₂₁Z₁C(O)R₂₂R₂₉Z₂C(O) where R₂₁ and R₂₂ are asdetailed above, Z₁, and Z₂ are each independently either NH, sulfur oroxygen, and R₂₉ is a C₁₋₅ alkyl, and the terminal carbonyl group isattached to the ring structure of the acceptor dye. In the variationwhere Z₂ is nitrogen, the C(O)R₂₂R₂₉Z₂ subunit forms an amino acidsubunit.

In this embodiment, the linker may be formed by the reaction of anactivated carbonyl group (NHS ester) with an amine, hydroxyl or thiolgroup. It is noted that a wide variety of other mechanisms for attachingan R₂₂ group to an acceptor dye are envisaged and are intended to fallwithin the scope of the invention.

Particular examples of five or six membered rings which may be used asR₂₂ in the linker include, but are not limited to cyclopentene,cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole,isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene,pyridine, pyridazine, pyrimidine, pyrazine and oxazine. Examples offused ring structures include, but are not limited to indene,benzofuran, thionaphthene, indole and naphthalene.

A preferred embodiment of this linker is where R₂₁ and R₂₉ aremethylene, Z₁, and Z₂ are NH, and R₂₂ is benzene, as shown below.

One class of energy transfer fluorescent dyes according to the presentinvention includes a donor dye which has the following xanthene ringstructure with a 4′ ring position:

where Y₁ and Y₂ taken separately are either hydroxyl, oxygen, iminium oramine, the iminium and amine preferably being a tertiary iminium oramine. R₁₁-R₁₇ may be any substituent which is compatible with theenergy transfer dyes of the present invention, it being noted that theR₁₁-R₁₇ may be widely varied in order to alter the spectral and mobilityproperties of the dyes.

According to this embodiment, the energy transfer dye also includes anacceptor dye which absorbs the excitation energy emitted by the donordye and fluoresces at a second wavelength in response. The energytransfer dye also includes a linker which attaches the donor dye to theacceptor dye.

In one variation of this embodiment of energy transfer dyes, the linkerhas the general structure R₂₁Z₁C(O)R₂₂R₂₈, as illustrated above, whereR₂₁ is a C₁₋₅ alkyl attached to the 4′ position of the xanthene donordye, C(O) is a carbonyl group, Z₁ is either NH, sulfur or oxygen, R₂₂ isa substituent attached to the carbonyl carbon which may be either analkene, diene, alkyne, a five or six membered ring having at least oneunsaturated bond or a fused ring structure, and R₂₈ includes afunctional group which attaches the linker to the acceptor dye.

In a further variation of this embodiment of energy transfer dyes, thelinker has the general structure R₂₁Z₁C(O)R₂₂R₂₉Z₂C(O), as illustratedabove, where R₂₁ and R₂₂ are as detailed above, Z₁ and Z₂ are eachindependently either NH, sulfur or oxygen, and R₂₉ is a C₁₋₅ alkyl, andthe terminal carbonyl group is attached to the ring structure of theacceptor dye. In the variation where Z₂ is nitrogen, —C(O)R₂₂R₂₉Z₂-formsan amino acid subunit.

In a further preferred variation of this embodiment of energy transferdyes, the linker is where R₂₁ and R₂₉ are methylene, Z₁ and Z₂ are NH,and R₂₂ is benzene, as shown below.

The donor dye may optionally be a member of the class of dyes where R₁₇is a phenyl or substituted phenyl. When Y₁ is hydroxyl and Y₂ is oxygen,and R₁₇ is a phenyl or substituted phenyl, the dye is a member of thefluorescein class of dyes. When Y₁ is amine and Y₂ is iminium, and R₁₇is a phenyl or substituted phenyl, the dye is a member of the rhodamineclass of dyes. Further according to this embodiment, the acceptor dyemay optionally be a member of the xanthene, cyanine, phthalocyanine andsquaraine classes of dyes.

In another embodiment, the energy transfer fluorescent dyes have donorand acceptor dyes with the general structure

where Y₁ and Y₂ taken separately are either hydroxyl, oxygen, iminium oramine, the iminium and amine preferably being a tertiary iminium oramine and R₁₁-R₁₆ are any substituents which are compatible with theenergy transfer dyes of the present invention.

According to this embodiment, as illustrated below, the linker isattached to one of X₃ and X₄ substituents of each of the donor andacceptor dyes, preferably the X₃ substituents of the donor and acceptordyes. In this embodiment, the linker is preferably short and/or rigid asthis has been found to enhance the transfer of energy between the donorand acceptor dyes.

In another embodiment, the energy transfer fluorescent dyes include adonor dye which is a member of the xanthene class of dyes, an acceptordye which is a member of the xanthene, cyanine, phthalocyanine andsquaraine classes of dyes which is capable of absorbing the excitationenergy emitted by the donor dye and fluorescing at a second wavelengthin response, and a linker attaching the donor dye to the acceptor dye.According to this embodiment, the acceptor has an emission maximum thatis greater than about 600 nm or at least about 100 nm greater than theabsorbance maximum of the donor dye.

In addition to the above-described novel energy transfer fluorescentdyes, the present invention also relates to fluorescent reagentscontaining the energy transfer fluorescent dyes. In general, thesereagents include any molecule or material to which the energy transferdyes of the invention can be attached and used to detect the presence ofthe reagent based on the fluorescence of the energy transfer dye. In oneembodiment, a fluorescent reagent is provided which includes anucleoside or a mono-, di- or triphosphate nucleotide labeled with anenergy transfer fluorescent dye. The nucleotide may be a deoxynucleotidewhich may be used for example, in the preparation of dye labeledoligonucleotides. The nucleotide may also be a dideoxynucleoside whichmay be used, for example, in dye terminator sequencing. In anotherembodiment, the fluorescent reagent includes an oligonucleotide labeledwith an energy transfer fluorescent dye. These reagents may be used, forexample, in dye primer sequencing.

The present invention also relates to methods which use the energytransfer fluorescent dyes and reagents of the present invention. In oneembodiment, the method includes forming a series of different sizedoligonucleotides labeled with an energy transfer fluorescent dye of thepresent invention, separating the series of labeled oligonucleotidesbased on size, detecting the separated labeled oligonucleotides based onthe fluorescence of the energy transfer dye.

In one embodiment of this method, a mixture of extended labeled primersis formed by hybridizing a nucleic acid sequence with an oligonucleotideprimer in the presence of deoxynucleotide triphosphates, and at leastone dye labeled dideoxynucleotide triphosphate and a DNA polymerase. TheDNA polymerase serves to extend the primer with the deoxynucleotidetriphosphates until a dideoxynucleotide triphosphate is incorporatedwhich terminates extension of the primer. Once terminated, the mixtureof extended primers are separated and detected based on the fluorescenceof the dye on the dideoxynucleoside. In a variation of this embodiment,four different fluorescently labeled dideoxynucleotide triphosphates areused, i.e., a fluorescently labeled dideoxycytosine triphosphate, afluorescently labeled dideoxyadenosine triphosphate, a fluorescentlylabeled dideoxyguanosine triphosphate, and a fluorescently labeleddideoxythymidine triphosphate. In an alternate embodiment of thismethod, the oligonucleotide primer is fluorescently labeled as opposedto the deoxynucleotide triphosphate.

The present invention also relates to kits containing the dyes andreagents for performing DNA sequencing using the dyes and reagents ofpresent invention.

5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the modification of a carboxy substituent on a energytransfer dye to an activated N-hydroxysuccinimidyl (NHS) ester which isthen reacted with an aminohexyl-oligomer to form a dye labeledoligonucleotide primer.

FIG. 2 compares the fluorescence emission strength of a series of energytransfer dyes of the present invention to other energy transfer dyes andthe acceptor dye alone.

FIGS. 3A and 3B show several particularly preferred embodiments of4,7-dichlororhodamine dye compounds which can be used in the energytransfer dyes of the present invention.

FIGS. 4A and 4B show preferred generalized synthesis schemes for thepreparation of the 4,7-dichlororhodamine dyes of the invention.

FIG. 4A shows a generalized synthesis wherein the substituent X₁ can beother than carboxylate.

FIG. 4B shows a generalized synthesis wherein the substituent X₁ iscarboxylate.

FIG. 5 illustrates a set of four dyes (3-carboxy-R110, 5-carboxy-R6G,5TMR-B-CF and 5ROX-CF) which are spectrally resolvable from each other.

FIG. 6 illustrates a set of four dyes (3-carboxy-R110, 5-carboxy-R6G,5ROX-CF and Cy5-CF) which are spectrally resolvable from each other.

FIG. 7 is a plot of a mixture of labeled oligonucleotides generatedduring dye primer sequencing using 5TMR-CF and 5TMR-B-CF labeledprimers.

FIG. 8 is a four color plot of dye primer sequencing using a four dyeset including 3-carboxy-R110, 5-carboxy-R6G, 5TMR-CF and 5TMR-B-CF.

FIGS. 9A-D compare the fluorescence emission strength of a series ofenergy transfer dyes of the present invention to the correspondingacceptor dye alone.

FIG. 9A provides the overlaid spectra of 6-CFB-DR110-2 and DR110-2.

FIG. 9B provides an overlaid spectra of 5-CFB-DR6G-2 and DR6G-2.

FIG. 9C provides an overlaid spectra of 6-CFB-DTMR-2 and DTMR-2.

FIG. 9D provides an overlaid spectra of 6-CFB-DROX-2 and DROX-2.

FIG. 10 illustrates a set of four dyes(5-CFB-DR110-2,5-CFB-DR6G-2,6-CFB-DTMR-2, and 6-CFB-DROX-2) which arespectrally resolvable from each other.

FIG. 11 is a plot of a mixture of labeled oligonucleotides generatedduring dye primer sequencing using 6-CFB-DTMR-2 and DTMR-2 labeledprimers.

FIG. 12 is a plot of a mixture of labeled oligonucleotides generatedduring dye primer sequencing using 5-CF-TMR-2 and 5-CF-B-TMR-2 labeledprimers.

FIG. 13 is a four color plot of dye primer sequencing using a four dyeset including 5-CFB-DR110-2,6-CFB-DR6g-2,5-CFB-DTMR-2, and 5-CFB-DROX-2.

6. DETAILED DESCRIPTION

6.1 Energy Transfer Dye Linkers of the Present Invention

The present invention relates to novel linkers for linking a donor dyeto an acceptor dye in an energy transfer fluorescent dye. The presentinvention also relates to energy transfer fluorescent dyes whichincorporate these linkers. These linkers have been found to faciliatethe efficient transfer of energy between a donor and acceptor dye in anenergy transfer dye.

One linker according to the present invention for linking a donor dye toan acceptor dye in an energy transfer fluorescent dye has the generalstructure R₂₁Z₁C(O)R₂₂R₂₈, as illustrated below, where R₂₁ is a C₁₋₅alkyl attached to the donor dye, C(O) is a carbonyl group, Z₁, is eitherNH, sulfur or oxygen, R₂₂ is a substituent which includes an alkene,diene, alkyne, a five and six membered ring having at least oneunsaturated bond or a fused ring structure which is attached to thecarbonyl carbon, and R₂₈ includes a functional group which attaches thelinker to the acceptor dye.

In one embodiment of this linker, illustrated below, the linker has thegeneral structure R₂₁Z₁C(O)R₂₂R₂₉Z₂C(O) where R₂₁ and R₂₂ are asdetailed above, Z₁, and Z₂ are each independently either NH, sulfur oroxygen, R₂₉ is a C₁₋₅ alkyl, and the terminal carbonyl group is attachedto the ring structure of the acceptor dye. In the variation where Z₂ isnitrogen, the C(O)R₂₂R₂₉Z₂ subunit forms an amino acid subunit.

Particular examples of five or six membered rings which may be used asR₂₂ in the linker include, but are not limited to cyclopentene,cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole,isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene,pyridine, pyridazine, pyrimidine, pyrazine and oxazine. Examples offused ring structures include, but are not limited to indene,benzofuran, thionaphthene, indole and naphthalene.

A preferred embodiment of this linker is where R₂₁ and R₂₉ aremethylene, Z₁, and Z₂ are NH, and R₂₂ is benzene, as shown below.

Table 3 illustrates examples of —C(O)R₂₂— subunits of linkers which maybe used in the linkers of the present invention.

6.2 Energy Transfer Dyes of the Present Invention

In general, the energy transfer dyes of the present invention include adonor dye which absorbs light at a first wavelength and emits excitationenergy in response, an acceptor dye which is capable of absorbing theexcitation energy emitted by the donor dye and fluorescing at a secondwavelength in response, and a linker which attaches the donor dye to theacceptor dye. With regard to all of the molecular structures providedherein, it is intended that these molecular structures encompass notonly the exact electronic structure presented, but also include allresonant structures and protonation states thereof.

One class of energy transfer fluorescent dyes according to the presentinvention includes a donor dye which is a member of the xanthene classof dyes, an acceptor dye and a linker which is a member of the group oflinkers described in Section 6.1 As used herein, xanthene dyes includeall molecules having the general structure:

where Y₁ and Y₂ taken separately are either hydroxyl, oxygen, iminium oramine, the iminium and amine preferably being a tertiary iminium oramine. When Y₁ is hydroxyl and Y₂ is oxygen, and R₁₇ is a phenyl orsubstituted phenyl, the dye is a member of the fluorescein class ofdyes. When Y₁ is amine and Y₂ is iminium, and R₁₇ is a phenyl orsubstituted phenyl, the dye is a member of the rhodamine class of dyes.

R₁₁-R₁₇ may be any substituent which is compatible with the energytransfer dyes of the present invention, it being noted that the R₁₁-R₁₇may be widely varied in order to alter the spectral and mobilityproperties of the dyes. The number indicated in the ring structureindicates the 4′ position on the xanthene ring structure. For the energytransfer dyes of the present invention in which the linker is attachedto the 4′ position of the xanthene ring structure, the R₁₄ substituentcorresponds to the linker.

Examples of R₁₁-R₁₇ substituents include, but not limited to hydrogen,fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne,sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substitutedphenyl, where adjacent substituents are taken together to form a ring,and combinations thereof.

In one embodiment, R₁₅ and R₁₆ are taken together to form a substitutedor unsubstituted benzene ring. This class of xanthene dyes are referredto herein as asymmetric benzoxanthene dyes and are described in U.S.application Ser. No. 08/626,085, filed Apr. 1, 1996, now U.S. Pat. No.6,020,481, entitled Asymmetric Benzoxanthene Dyes, by Scott C. Benson etal. which is incorporated herein by reference.

In another embodiment, R₁₇ is a phenyl or substituted phenyl having thegeneral formula

Substituents X₁-X₅ on the phenyl ring can include hydrogen, fluorine,chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate,amino, ammonium, amido, nitrile, alkoxy, where adjacent substituents aretaken together to form a ring, and combinations thereof.

In one embodiment, the donor dye is a member of the class of dyes whereY₁ is amine, Y₂ is iminium, and X₂ and X₅ are chlorine, referred toherein as 4,7-dichlororhodamine dyes. Dyes falling within the4,7-dichlororhodamine class of dyes and their synthesis are describedherein as well as in U.S. application Ser. No. 08/672,196, filed Jun.27, 1996, now U.S. Pat. No. 5,847,162, entitled “4,7-DICHLORORHODAMINEDYES,” which is incorporated herein by reference.

As used here, alkyl denotes straight-chain and branched hydrocarbonmoieties, i.e., methyl, ethyl, propyl, isopropyl, tert-butyl, isobutyl,sec-butyl, neopentyl, tert-pentyl, and the like. Substituted alkyldenotes an alkyl moiety substituted with any one of a variety ofsubstituents, including, but not limited to hydroxy, amino, thio, cyano,nitro, sulfo, and the like. Haloalkyl denotes a substituted alkyl withone or more halogen atom substituents, usually fluoro, chloro, bromo, oriodo. Alkene denotes a hydrocarbon wherein one or more of thecarbon-carbon bonds are double bonds, and the non-double bonded carbonsare alkyl or substituted alkyl. Alkyne denotes a hydrocarbon where oneor more of the carbons are bonded with a triple bond and where thenon-triple bonded carbons are alkyl or substituted alkyl moieties.Sulfonate refers to moieties including a sulfur atom bonded to 3 oxygenatoms, including mono- and di-salts thereof, e.g., sodium sulfonate,potassium sulfonate, disodium sulfonate, and the like. Amino refers tomoieties including a nitrogen atom bonded to 2 hydrogen atoms, alkylmoieties, or any combination thereof. Amido refers to moieties includinga carbon atom double bonded to an oxygen atom and single bonded to anamino moiety. Nitrile refers to moieties including a carbon atom triplebonded to a nitrogen atom. Alkoxy refers to a moiety including an alkylmoiety single bonded to an oxygen atom. Aryl refers to single ormultiple phenyl or substituted phenyl, e.g., benzene, naphthalene,anthracene, biphenyl, and the like.

R₁₁-R₁₇ may also each independently be a linking moiety which may beused to attach the energy transfer dye to a reagent, such as anucleotide, nucleoside or oligonucleotide. Examples of linking moietiesinclude isothiocyanate, sulfonyl chloride, 4,6-dichlorotriazinylamine,succinimidyl ester, or other active carboxylate whenever thecomplementary functionality is amine. Preferably the linking group ismaleimide, halo acetyl, or iodoacetamide whenever the complementaryfunctionality is sulfhydryl. See R. Haugland, Molecular Probes Handbookof Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.(1992). In a particularly preferred embodiment, as illustrated in FIG.1, the linking group is an activated NHS ester formed from a carboxylgroup on either the donor or acceptor dye which can be reacted with anaminohexyl-oligomer to form a dye labeled oligonucleotide primer.

The energy transfer fluorescent dyes of this embodiment also include anacceptor dye which is capable of absorbing the excitation energy emittedby the donor dye and fluorescing at a second wavelength in response, anda linker which attaches the donor dye to the acceptor dye. In the firstclass of energy transfer dyes, the linker is a member of the class oflinkers described in Section I and is attached to the donor dye at the4′ position of the xanthene ring structure.

Energy transfer dyes of this first class exhibit enhanced fluorescentstrength as compared to the acceptor fluorophore itself and energytransfer fluorescent dyes having the same donor-acceptor pair where thelinkage between the donor-acceptor pair is different.

The present invention also relates to a second class of energy transferfluorescent dyes in which the donor and acceptor dyes each have thegeneral structure

where Y₁, Y₂, R₁₁-R₁₆ and X₁-X₅ are as specified above.

Within this class of dyes, the linker is attached to the donor andacceptor dyes by one of X₃ and X₄ substituents of each of the donor andacceptor dyes.

In a preferred embodiment of this class of dyes, the linker is attachedto the donor and acceptor dyes by the X₃ substituent of each of thedonor and acceptor dyes.

Within this class of dyes, the linker is preferably short and/or rigidas this has been found to enhance the transfer of energy between thedonor and acceptor dyes.

The present invention also relates to a third class of energy transferfluorescent dyes in which the acceptor dye is a member of the4,7-dichlororhodamine class of dyes, i.e., dyes having the generalstructure

where:

-   -   R₁-R₄ are each independently hydrogen, alkyl or where R₁ and R₅,        R₂ and R₆, R₃ and R₈, R₄ and R₉ are taken together to form a        ring, and combinations thereof;    -   R₅-R₁₀ are each independently hydrogen, fluorine, chlorine,        bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate,        sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl, or        substituted phenyl, or where adjacent substituents are taken        together to form a ring, and combinations thereof;    -   X₁, X₃ and X₄ are each independently hydrogen, fluorine,        chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne,        sulfonate, sulfone, amino, ammonium, amido, nitrile, or alkoxy,        or where adjacent substituents are taken together to form a        ring, and combinations thereof; and    -   X₂ and X₅ are chlorine.

With regard to R₁-R₁₀, X₃ and X₄, R₁ and R₅, R₂ and R₆, R₃ and R₈, R₄and R₉, and X₃ and X₄ may each independently be taken together to form a5, 6, or 7 membered ring.

The numbers (4′, 5, 6) indicated in the ring structure indicate the 4′-,5- and 6-ring positions on the rhodamine ring structure. As will bediscussed herein, the 4′- and 5-ring positions are preferred sites forattachment of the linker used in the energy transfer dyes of the presentinvention which attaches the donor to the acceptor fluorophore. The 4′-,5- and 6-ring positions are also preferred sites for attachment of abiomolecule, such as a nucleotide or oligonucleotide to the energytransfer dye.

Donor dyes within this class of energy transfer dyes may include any dyewhich emits excitation energy which a 4,7-dichlororhodamine dye iscapable of absorbing and producing an energy emission in response. Inone embodiment, the donor dye has a xanthene ring structure with a 4′ring position where the 4,7-dichlororhodamine acceptor dye is attachedto the donor dye by a linker which is attached to the 4′ ring positionof the xanthene dye. The linker is preferably attached to the 5 or 6ring positions of the 4,7-dichlororhodamine acceptor dye.

Energy transfer dyes according to this third class of dyes, i.e., where4,7-dichlororhodamine is the acceptor dye, provide the advantage ofhaving a relatively narrow emission spectrum as compared to otherrhodamine dyes. This narrow emission spectrum enhances the spectralresolution achievable by a set of these dyes, thereby facilitatingmulticomponent analysis using these dyes.

The present invention also relates to a fourth class of energy transferfluorescent dyes in which the donor dye is a member of the xantheneclass of dyes, the acceptor dye is a member of the xanthene, cyanine,phthalocyanine and squaraine classes of dyes, and the acceptor has anemission maximum that is greater than about 600 nm and/or preferably hasan emission maximum that is at least about 100 nm greater than theabsorbance maximum of the donor dye. Within this class of dyes, thedonor is preferably a member of the fluorescein class of dyes.

The fourth class of energy transfer dyes of the present inventionexhibit unusually large Stoke shifts, as measured by the differencebetween the absorbance of the donor and the emission of the acceptor. Inaddition, these dyes exhibit efficient energy transfer in that minimaldonor fluorescence is observed.

Described herein in greater detail are the four classes of energytransfer dyes of the present invention.

TABLE 1

5-CFB-DR110-2

5-CFB-DR6G-2

6-CFB-DR110-2

6-CFB-DR6G-2

5-CFB-DTMR-2

5-CFB-DROX-2

6-CFB-DTMR-2

6-CFB-DROX-2

TABLE 1A

6.2.1 First Class of Energy Transfer Dyes

As described above, the first class of energy transfer dyes according tothe present invention includes a donor dye which is a member of thexanthene class of dyes and hence has a xanthene ring structure with a 4′ring position. Within this class of dyes, the acceptor dye is a dyewhich is capable of absorbing the excitation energy emitted by the donordye and fluorescing at a second wavelength in response.

According to this embodiment, the donor may be a member of thefluorescein, rhodamine or asymmetric benzoxanthene classes of dyes,these dyes each being members of the broader xanthene class of dyes.Illustrated below are the general structural formulas for these xanthenedyes. The substituents illustrated on these dyes may be selected fromthe wide variety of substituents which may be incorporated onto thesedifferent classes of dyes since all dyes having the general xanthene,fluorescein, rhodamine, and asymmetric benzoxanthene ring structures areintended to fall within the scope of this invention.

Examples of classes of acceptor dyes which may be used in the energytransfer fluorescent dye of this embodiment include, but are not limitedto, xanthene dyes, cyanine dyes, phthalocyanine dyes and squaraine dyes.The general structures of these dyes are illustrated in Table 1A. Thesubstituents illustrated on these dyes may be selected from the widevariety of substituents which may be incorporated onto these differentclasses of dyes since all dyes having the general xanthene, fluorescein,rhodamine, asymmetric benzoxanthene, cyanine, phthalocyanine andsquaraine ring structures are intended to fall within the scope of thisinvention.

Examples of donor dyes which may be used in this embodiment include, butare not limited to fluorescein, isomers of carboxyfluorescein (e.g., 5and 6 carboxy), isomers of carboxy-HEX (e.g., 5 and 6 carboxy), NAN,Cl-FLAN, TET, JOE, ZOE, rhodamine, isomers of carboxyrhodamine (e.g., 5and 6 carboxy), isomers of carboxy R110 (e.g., 5 and 6 carboxy), isomersof carboxy R6G (e.g., 5 and 6 carboxy), 4,7-dichlorofluoresceins (SeeU.S. Pat. No. 5,188,934), 4,7-dichlororhodamines (See application Ser.No. 08/672,196, filed Jun. 27, 1996, now U.S. Pat. No. 5,847,162),asymmetric benzoxanthene dyes (See U.S. application Ser. No. 08/626,085,filed Apr. 1, 1996, now U.S. Pat. No. 6,020,481), and isomers ofN,N,N′,N′-tetramethyl-carboxyrhodamine (TAMRA) (e.g., 5 and 6 carboxy).

Examples of acceptor dyes which may be used in this embodiment include,but are not limited to isomers of carboxyfluorescein (e.g., 5 and 6carboxy), 4,7-dichlorofluoresceins, 4,7-dichlororhodamines,fluoresceins, asymmetric benzoxanthene dyes, isomers of carboxy-HEX(e.g., 5 and 6 carboxy), NAN, Cl-FLAN, TET, JOE, ZOE, rhodamine, isomersof carboxyrhodamine (e.g., 5 and 6 carboxy), isomers of carboxy R110(e.g., 5 and 6 carboxy), isomers of carboxy R6G (e.g., 5 and 6 carboxy),isomers of N,N,N′,N′-tetramethyl carboxyrhodamine (TAMRA) (e.g., 5 and 6carboxy), isomers of carboxy-X-rhodamine (ROX) (e.g., 5 and 6 carboxy)and Cy5. Illustrated in Table 2 are the structures of these dyes.

In the first class of energy transfer dyes according to the presentinvention, the linker is attached to the donor dye at the 4′ position ofthe xanthene ring structure. In one embodiment, the linker has thegeneral structure R₂₁Z₁C(O)R₂₂R₂₈, as illustrated below, where R₂₁ is aC₁₋₅ alkyl which is attached to the 4′ ring position of the donorxanthene dye, Z₁ is either NH, sulfur or oxygen, C(O) is a carbonylgroup, R₂₂ is a substituent which includes an alkene, diene, alkyne, afive and six membered ring having at least one unsaturated bond or afused ring structure which is attached to the carbonyl carbon, and R₂₈is a functional group which attaches the linker to the acceptor dye.

Examples of five or six membered rings which may be used in R₂₂ include,but are not limited to cyclopentene, cyclohexene, cyclopentadiene,cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole,pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine,pyrimidine, pyrazine and oxazine. Examples of fused ring structuresinclude, but are not limited to indene, benzofuran, thionaphthene,indole and naphthalene.

In one variation of this embodiment, illustrated below, the linker hasthe general structure R₂₁Z₁C(O)R₂₂R₂₉Z₂C(O) where R₂₁ is a C₁₋₅ alkylwhich is attached to the 4′ ring position of the donor xanthene dye, Z₁,and Z₂ are each independently either NH, sulfur or oxygen, C(O) is acarbonyl group, R₂₂ is a substituent which includes an alkene, diene,alkyne, a five and six membered ring having at least one unsaturatedbond or a fused ring structure which is attached to the carbonyl carbon,R₂₉ is a C₁₋₅ alkyl, and the terminal carbonyl group is attached to thering structure of the acceptor dye.

A preferred embodiment of this linker is where R₂₁ and R₂₉ aremethylene, Z₁, and Z₂ are NH, and R₂₂ is benzene, as shown below.

TABLE 2

TABLE 3

As illustrated in Example 4 and FIG. 2, energy transfer dyes such as5-TMR-B-CF, which include a donor, acceptor and linker as specifiedabove exhibit enhanced fluorescence as compared to the acceptor itselfand energy transfer fluorescent dyes having the same donor-acceptor pairwhere the linker between the donor-acceptor pair is different. Withoutbeing bound by theory, the enhanced fluorescence intensity observed isbelieved to be due to an improved energy transfer orientation betweenthe donor and acceptor dye which is achieved and maintained by therelatively rigid R₂₂ portion of the linker. As a result, the energytransfer fluorescent dyes of the present invention exhibit enhancedfluorescent strength as compared to the acceptor fluorophore itself andenergy transfer fluorescent dyes having the same donor-acceptor pairwhere the linkage between the donor-acceptor pair is different. Theenhanced fluorescent strength of these dyes is particularly evident inthe presence of 8 M urea which serves to reduce dye stacking.

In one variation of this embodiment, the acceptor is a member of thexanthene class of dyes having the general structure

where Y₁, Y₂, R₁₁-R₁₆ and X₁-X₅ are as specified above.

According to this variation, it is preferred that a linker, such as theones described above, is attached to the acceptor xanthene dye via theX₃ or X₄ substituent of the acceptor xanthene dye. In a preferredembodiment, as illustrated below, the linker is attached to the X₃substituent of the acceptor xanthene dye.

Table 4 provides examples of the above-described energy transfer dyesaccording to this embodiment of the invention. It is noted that althoughthe dyes illustrated in Table 4 include a 5-carboxyfluorescein donor dyeand a TAMRA acceptor dye, it should be understood that a wide variety ofother xanthene dyes can be readily substituted as the donor dye. Itshould also be understood that a wide variety of other xanthene dyes, aswell as cyanine, phthalocyanine and squaraine dyes can be readilysubstituted for the TAMRA acceptor dye, as has been described above, allof these variations with regard to the donor and acceptor dyes fallingwithin the scope of the invention.

TABLE 4

5-TMR-B-CF

5-TMR-F-CF

5-TMR-P-CF

5-TMR-A-CF

5-TMR-D-CF

5-TMR-N-CF

5-DTMR-B-CF

5-DTMR-F-CF

5-DTMR-P-CF

5-DTMR-A-CF

5-DTMR-D-CF

5-DTMR-N-CF

6.2.2 Second Class of Energy Transfer Dyes

The present invention also relates to a second class of energy transferfluorescent dyes, illustrated below, in which the donor dye and acceptoreach are members of the xanthene class of dyes having the generalstructure

where Y₁, Y₂, R₁₁-R₁₆ and X₁-X₅ are as specified above.

According to this embodiment, the linker is attached to the X₃ or X₄substituent of both the donor and acceptor dyes, as illustrated below.

In this embodiment, the linker is preferably short and/or rigid as thishas been found to enhance the transfer of energy between the donor andacceptor dyes. For example, in one variation of this embodiment, thelinker preferably has a backbone attaching the donor to the acceptorwhich is less than 9 atoms in length. In another variation of thisembodiment, the linker includes a functional group which gives thelinker some degree of structural rigidity, such as an alkene, diene, analkyne, a five and six membered ring having at least one unsaturatedbond or a fused ring structure. In yet another variation, the linker hasthe general formula R₂₅Z₃C(O) or R₂₅Z₃C(O)R₂₆Z₄C(O) where R₂₅ isattached to the donor dye, C(O) is a carbonyl group and the terminalcarbonyl group is attached to the acceptor dye, R₂₅ and R₂₆ are eachselected from the group of C₁₋₄ alkyl, and Z₃ and Z₄ are eachindependently either NH, O or S.

Examples of donor and acceptor dyes which may be used in this embodimentinclude, but are not limited to fluorescein, 5 or 6 carboxyfluorescein,5 or 6 carboxy-HEX, NAN, Cl-FLAN, TET, JOE, ZOE,4,7-dichlorofluoresceins, asymmetric benzoxanthene dyes, rhodamine, 5 or6 carboxyrhodamine, 5 or 6 carboxy-R110, 5 or 6 carboxy-R6G,N,N,N′,N′-tetramethyl (5 or 6)-carboxyrhodamine (TAMRA), 5 or 6carboxy-X-rhodamine (ROX) and 4,7-dichlororhodamines. Illustrated inTable 2 are the structures of these dyes.

In another variation of this embodiment, the linker includes a R₂₇Z₅C(O)group where R₂₇ is a C₁₋₅ alkyl attached to the donor dye, Z₅ is eitherNH, sulfur or oxygen, and C(O) is a carbonyl group attached to theacceptor dye.

Table 5 provides examples of the second class of energy transfer dyesaccording to the present invention. It is noted that although the dyesillustrated in Table 5 include a 5-aminomethylfluorescein donor dye, itshould be understood that a wide variety of other xanthene dyes can bereadily substituted as the donor dye. It should also be understood thata wide variety of other xanthene dyes, as well as cyanine,phthalocyanine and squaraine dyes can be readily substituted for theTAMRA acceptor dye, as has been described above, all of these variationswith regard to the donor and acceptor dyes falling within the scope ofthe invention.

TABLE 5

6.2.3 Third Class of Energy Transfer Dyes

The third class of energy transfer fluorescent dyes include a4,7-dichlororhodamine dye as the acceptor dye and a dye which producesan emission which the 4,7-dichlororhodamine dye can absorb as the donordye. These dyes exhibit enhanced fluorescence intensity as compared tothe acceptor dye alone. In addition, 4,7-dichlororhodamine dyes exhibita narrower emission spectrum than other rhodamine dyes which facilitatestheir use in multiple component analyses.

In a preferred embodiment, these energy transfer dyes include those dyesaccording to the first and second classes of dyes in which the acceptoris a 4,7-dichlororhodamine dye.

6.2.3.1 4,7-Dichlororhodamine Dyes

4,7-dichlororhodamine dye compounds have the general structure

where:

-   -   R₁-R₄ are each independently hydrogen, alkyl or where R₁ and R₅,        R₂ and R₆, R₃ and R₈, R₄ and R₉ are taken together to form a        ring, and combinations thereof;    -   R₅-R₁₀ are each independently hydrogen, fluorine, chlorine,        bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate,        sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl, or        substituted phenyl, or where adjacent substituents are taken        together to form a ring, and combinations thereof;    -   X₁, X₃ and X₄ are each independently hydrogen, fluorine,        chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne,        sulfonate, sulfone, amino, ammonium, amido, nitrile, or alkoxy,        or where adjacent substituents are taken together to form a        ring, and combinations thereof; and    -   X₂ and X₅ are chlorine.

Dyes falling within the 4,7-dichlororhodamine class of dyes and theirsynthesis are described in U.S. application Ser. No. 08/672,196, filedJun. 27, 1996, now U.S. Pat. No. 5,847,162, entitled“4,7-DICHLORORHODAMINE DYES,” which is incorporated herein by reference.

With regard to R₁-R₄, alkyl substituents may include between about 1 to8 carbon atoms (i.e., methyl, ethyl, propyl, isopropyl, tert-butyl,isobutyl, sec-butyl, neopentyl, tert-pentyl, and the like) and may bestraight-chain and branched hydrocarbon moieties. In a preferredembodiment, R₁-R₄ are each independently either hydrogen, methyl, orethyl and more preferably either hydrogen or methyl.

With regard to R₅-R₁₀, alkyl, alkene, alkyne and alkoxy substituentspreferably include between about 1 to 8 carbon atoms (i.e., methyl,ethyl, propyl, isopropyl, tert-butyl, isobutyl, sec-butyl, neopentyl,tert-pentyl, and the like) and may be straight-chain and branchedhydrocarbon moieties.

With regard to R₁-R₁₀, R₁ and R₅, R₂ and R₆, R₃ and R₈, R₄ and R₉ mayeach independently be taken together to form a 5, 6, or 7 membered ring.

In one embodiment, R₆ and R₇ is benzo, and/or, R₉ and R₁₀ is benzo. In apreferred embodiment, R₅-R₁₀ are each independently either hydrogen,methyl, or ethyl and more preferably either hydrogen or methyl.

With regard to X₁, X₃ and X₄, X₁ is preferably a carboxylate and one ofX₃ and X₄ may include a substituent which is used to link the4,7-dichlororhodamine acceptor dye to a donor dye or to link anucleotide or an oligonucleotide to the energy transfer dye. The R₈substituent at the 4′ ring position may also be used to link theacceptor to either the donor dye or to a biomolecule such as anucleotide or oligonucleotide.

In one particularly preferred acceptor dye that may be used in thepresent invention, referred to herein as DR110-2, R₁-R₁₀ takenseparately are hydrogen, X₁ is carboxylate, and one of X₃ and X₄ is alinking group (L), the other being hydrogen. The structure of DR110-2 isshown below.

In a second particularly preferred acceptor dye that may be used in thepresent invention, referred to herein as DR6G-2, one of R₁ and R₂ isethyl, the other being hydrogen, one of R₃ and R₄ is ethyl, the otherbeing hydrogen, R₅ and R₈ taken separately are methyl, R₆, R₇, R₉, andR₁₀ are hydrogen, X₁ is carboxylate, and one of X₃ and X₄ is a linkinggroup, the other being hydrogen. The structure of DR6G-2 is shown below.

In a third particularly preferred acceptor dye that may be used in thepresent invention, referred to herein as DTMR, R₁-R₆ taken separatelyare hydrogen, Y₁-Y₄ taken separately are methyl, X₁ is carboxylate, andone of X₂ and X₃ is linking group, the other being hydrogen. Thestructure of DTMR is shown below.

In a fourth particularly preferred acceptor dye that may be used in thepresent invention, referred to herein as DROX, R₁ and R₆ are takentogether to form a six membered ring, R₂ and R₅ are taken together toform a six membered ring, R₃ and R₇ are taken together to form a sixmembered ring, R₄ and R₈ are taken together to form a six membered ring,R₅ and R₆ are hydrogen, X₁ is carboxylate, and one of X₃ and X₄ is alinking group, the other being hydrogen. The structure of DROX is shownbelow.

FIGS. 3A and 3B show several additional preferred embodiments of4,7-dichlororhodamine dyes which can be used in the energy transfer dyesof the present invention.

In compound 3A-A, one of R₁ and R₂ is ethyl, the other being hydrogen,R₃ and R₄ taken separately are hydrogen, R₆ is methyl, R₅ and R₇-R₁₀taken separately are hydrogen, X₁ is carboxylate, and one of X₃ and X₄is a linking group, the other being hydrogen.

In compound 3A-B, one of R₁ and R₂ is ethyl, the other being hydrogen,R₃ and R₄ taken separately are methyl, R₅ is methyl, R₆-R₁₀ takenseparately are hydrogen, X₁ is carboxylate, and, one of X₃ and X₄ is alinking group, the other being hydrogen.

In compound 3A-C, R₁ and R₂ taken separately are methyl, R₃ and R₉ takentogether form a six membered ring, R₄ and R₈ taken together form a sixmembered ring, R₅, R₆, R₇, and R₁₀ taken separately are hydrogen, X₁ iscarboxylate, and, one of X₃ and X₄ is a linking group, the other beinghydrogen.

In compound 3B-D, R₁ and R₂ taken separately are hydrogen, R₃ and R₉taken together form a six membered ring, R₄ and R₈ taken together form asix membered ring, R₅, R₆, R₇, and R₁₀ taken separately are hydrogen, X₁is carboxylate, and one of X₃ and X₄ is a linking group, the other beinghydrogen.

In compound 3B-E, one of R₁ and R₂ is ethyl, the other being hydrogen,R₃ and R₉ taken together form a six membered ring, R₄ and R₈ takentogether form a six membered ring, R₅ is methyl, R₆, R₇ and R₁₀ takenseparately are hydrogen, X₁ is carboxylate, and, one of X₃ and X₄ is alinking group, the other being hydrogen.

In compound 3B-F, R₁ and R₂ taken separately are hydrogen, R₃ and R₄taken separately are methyl, R₅-R₁₀ taken separately are hydrogen, X₁ iscarboxylate, and, one of X₃ and X₄ is linking group, the other beinghydrogen.

FIGS. 4A and 4B show preferred generalized synthesis schemes for thepreparation of 4,7-dichlororhodamine dyes used in the energy transferdyes of this invention. The variable substituents indicated in eachfigure are as previously defined.

FIG. 4A shows a generalized synthesis wherein the substituent X₁ can beother than carboxylate. In the figure, X′ indicates moieties which areprecursors to X₁. In the method illustrated in FIG. 4A, two equivalentsof a 3-aminophenol derivative 4A-A/4A-B, such as 3-dimethylaminophenol,is reacted with one equivalent of a dichlorobenzene derivative 4A-C,e.g., 4-carboxy-3,6, dichloro-2-sulfobenzoic acid cyclic anhydride,i.e., where the X₁′ moieties of 4A-C taken together are —C(O)—O—S(O)₂—.

The reactants are then heated for 12 h in a strong acid, e.g.,polyphosphoric acid or sulfuric acid, at 180° C. The crude dye 4A-D isprecipitated by addition to water and isolated by centrifugation. Toform a symmetrical product, the substituents of reactants 4A-A and 4A-Bare the same, while to form an asymmetrical product, the substituentsare different.

FIG. 4B shows a generalized synthesis wherein the substituent X₁ iscarboxylate. In the method of FIG. 4B, two equivalents of a3-aminophenol derivative 4A-A/4A-B, such as 3-dimethylaminophenol, isreacted with one equivalent of a phthalic anhydride derivative 4B-E,e.g. 3,6-dichlorotrimellitic acid anhydride. The reactants are thenheated for 12 h in a strong acid, e.g., polyphosphoric acid or sulfuricacid, at 180° C. The crude dye 4A-D is precipitated by addition to waterand isolated by centrifugation. To form a symmetrical product, thesubstituents of reactants 4A-A and 4A-B are the same, while to form anasymmetrical product, the substituents are different.

6.2.3.2 Energy Transfer Dyes with 4,7-Dichlororhodamine as the Acceptor

In general, the energy transfer dyes of the present invention include adonor dye which absorbs light at a first wavelength and emits excitationenergy in response, a 4,7-dichlororhodamine acceptor dye which iscapable of absorbing the excitation energy emitted by the donor dye andfluorescing at a second wavelength in response, and a linker whichattaches the donor dye to the acceptor dye. Preferred examples of thisclass of dyes which use a 4,7-dichlororhodamine dye as the acceptor dyeis illustrated in Table 1.

Examples of acceptor dyes which may be used in this class of dyesinclude, but are not limited to DR110-2, DR6G-2, DTMR, DROX, asillustrated above, as well as the dyes illustrated in FIGS. 3A-3B.

One subclass of these energy transfer fluorescent dyes are the dyesaccording to the first class of dyes of the present invention in whichthe acceptor dye is a 4,7-dichlororhodamine dye. The general structureof these dyes is illustrated below.

Table 4 provides examples of the energy transfer dyes belonging to thefirst class of dyes in which a 4,7 dichlororhodamine is used as theacceptor dye. It is noted that although the dyes illustrated in Table 4include a 5-carboxyfluorescein donor dye and a 5 or 6 carboxy DTMR asthe acceptor dye, it should be understood that a wide variety of otherxanthene dyes can be readily substituted as the donor dye and a widevariety of other 4,7-dichlororhodamine dyes can be readily substitutedfor the DTMR acceptor dye, all of these variations with regard to thedonor and acceptor dyes being intended to fall within the scope of theinvention.

Another subclass of these energy transfer fluorescent dyes are the dyesaccording to the second class of dyes of the present invention in whichthe acceptor dye is a 4,7-dichlororhodamine dye. The general structureof these dyes where the donor xanthene dye and acceptor4,7-dichlororhodamine dye are linked to each other at either the five orsix ring positions of the donor and acceptor dyes is illustrated below.

As described above, in this embodiment, the linker attaching the donorto the acceptor dye is preferably short and/or rigid as this has beenfound to enhance the transfer of energy between the donor and acceptordyes. The substituent labels shown above correspond to the same groupsof substituents as has been specified with regard to the other dyes.

Table 5 provides examples of the second class of energy transfer dyesaccording to the present invention in which 4,7 dichlororhodamine isused as the acceptor dye. It is noted that although the dyes illustratedin Table 5 include a 5-aminomethylfluorescein donor dye, it should beunderstood that a wide variety of other xanthene dyes can be readilysubstituted as the donor dye. It should also be understood that a widevariety of other 4,7-dichlororhodamine dyes can be readily substitutedfor the acceptor dye shown in Table 5 since, as has been describedabove, all of these variations with regard to the donor and acceptordyes are intended to fall within the scope of the invention.

6.2.4 Fourth Class of Energy Transfer Dyes

The present invention also relates to a fourth class of energy transferfluorescent dyes in which the donor dye is a member of the xantheneclass of dyes, and the acceptor dye is a member of the xanthene,cyanine, phthalocyanine or squaraine classes of dyes. Within this classof energy transfer dyes, it is preferred that the donor be a member ofthe fluorescein class of dyes and the acceptor dye have an emissionmaximum that is greater than about 600 nm and/or an emission maximumthat is at least about 100 nm greater than the absorbance maximum of thedonor dye.

The fourth class of dyes of the present invention exhibit unusuallylarge Stoke shifts, as measured by the difference between the absorbanceof the donor and the emission of the acceptor. In addition, these dyesexhibit efficient energy transfer in that minimal donor fluorescence isobserved. Interestingly, energy is transferred from the donor to theacceptor in some of the dyes belonging to this class even though theabsorbance spectrum of the acceptor dye does not overlap with theemission spectrum of the donor dye.

Examples of acceptor dyes which may be used in this embodiment include,but are not limited to 5-carboxy-X-rhodamine (ROX) and Cy5.

The energy transfer dyes of this embodiment also include a linker whichattaches the donor to the acceptor. The linker used to attach the donorto the acceptor dye may be any linker according to the first and secondclasses of dyes. However, it is foreseen that alternate linkers may beused in this class of dyes.

In one embodiment of this class of dyes, the linker is attached to the4′ position of the donor dye's xanthene ring structure. The linkerpreferably has a general structure R₂₁Z₁C(O)R₂₂R₂₈, as described abovewhere R₂₁ is a C₁₋₅ alkyl which is attached to the 4′ ring position ofthe donor xanthene dye, Z₁, is either NH, sulfur or oxygen, C(O) is acarbonyl group, R₂₂ is a substituent which includes an alkene, diene,alkyne, a five and six membered ring having at least one unsaturatedbond or a fused ring structure which is attached to the carbonyl carbon,and R₂₈ is a functional group which attaches the linker to the acceptordye. In cases where the acceptor dye is a member of the xanthene classof dyes, the linker is preferably attached to acceptor at the 5 positionof the xanthene ring structure.

Table 6 provides examples of the above-described energy transfer dyesaccording to the present invention. It is noted that although the dyesillustrated in Table 6 include a 5-carboxyfluorescein donor dye itshould be understood that a wide variety of other xanthene dyes can bereadily substituted as the donor dye. It should also be understood thata wide variety of other xanthene dyes, as well as cyanine,phthalocyanine and squaraine dyes can be readily substituted for the5-carboxy ROX and Cy5 acceptor dyes, as has been described above, all ofthese variations with regard to the donor and acceptor dyes fallingwithin the scope of the invention.

The energy transfer dyes of this embodiment exhibit unusually largeStoke shifts which make these dyes particularly well suited for use withdyes having smaller Stoke shifts in four dye DNA sequencing. Forexample, FIGS. 5 and 6 illustrate two sets of four dyes which arespectrally resolvable from each other. Within FIG. 5, 5ROX-CF is a dyefalling within the scope of the fourth class of dyes described above.Meanwhile, FIG. 6 includes 5ROX-CF and Cy5-CF which both fall within thescope of the fourth class of dyes described above.

As can be seen from the emission spectra of 5ROX-CF and Cy5-CFillustrated in FIG. 6, very little fluorescence from the donor dye(5-carboxyfluorescein, 520 nm) is observed in these dyes. This is anunexpected result in view of the large difference between the emissionmaximum of the donor dye (fluorescein) and the absorbance maximum of theacceptor dyes (ROX, 590 nm, Cy5, 640 nm).

TABLE 6

6.3 Reagents Including Energy Transfer Dyes of the Present Invention

The present invention also relates to fluorescent reagents whichincorporate an energy transfer fluorescent dye according to the presentinvention. As described in greater detail in Section III, these reagentsmay be used in a wide variety of methods for detecting the presence of acomponent in a sample.

The fluorescent reagents of the present invention include any moleculeor material to which the energy transfer dyes of the invention can beattached and used to detect the presence of the reagent based on thefluorescence of the energy transfer dye. Types of molecules andmaterials to which the dyes of the present invention may be attached toform a reagent include, but are not limited to proteins, polypeptides,polysaccharides, nucleotides, nucleosides, oligonucleotides,oligonucleotide analogs (such as a peptide nucleic acid), lipids, solidsupports, organic and inorganic polymers, and combinations andassemblages thereof, such as chromosomes, nuclei, living cells, such asbacteria, other microorganisms, mammalian cells, and tissues.

Preferred classes of reagents of the present invention are nucleotides,nucleosides, oligonucleotides and oligonucleotide analogs which havebeen modified to include an energy transfer dye of the invention.Examples of uses for nucleotide and nucleoside reagents include, but arenot limited to, labeling oligonucleotides formed by enzymatic synthesis,e.g., nucleoside triphosphates used in the context of PCR amplification,Sanger-type oligonucleotide sequencing, and nick-translation reactions.Examples of uses for oligonucleotide reagents include, but are notlimited to, as DNA sequencing primers, PCR primers, oligonucleotidehybridization probes, and the like.

One particular embodiment of the reagents are labeled nucleosides (NTP),such as cytosine, adenosine, guanosine, and thymidine, labeled with anenergy transfer fluorescent dye of the present invention. These reagentsmay be used in a wide variety of methods involving oligonucleotidesynthesis. Another related embodiment are labeled nucleotides, e.g.,mono-, di- and triphosphate nucleoside phosphate esters. These reagentsinclude, in particular, deoxynucleoside triphosphates (dNTP), such asdeoxycytosine triphosphate, deoxyadenosine triphosphate, deoxyguanosinetriphosphate, and deoxythymidine triphosphate, labeled with an energytransfer fluorescent dye of the present invention. These reagents may beused, for example, as polymerase substrates in the preparation of dyelabeled oligonucleotides. These reagents also include labeleddideoxynucleoside triphosphates (ddNTP), such as dideoxycytosinetriphosphate, dideoxyadenosine triphosphate, dideoxyguanosinetriphosphate, and dideoxythymidine triphosphate, labeled with an energytransfer fluorescent dye of the present invention. These reagents may beused, for example, in dye termination sequencing.

Another embodiment of reagents are oligonucleotides which includes anenergy transfer fluorescent dye of the present invention. These reagentsmay be used, for example, in dye primer sequencing.

As used herein, “nucleoside” refers to a compound consisting of apurine, deazapurine, or pyrimidine nucleoside base, e.g., adenine,guanine, cytosine, uracil, thymine, deazaadenine, deazaguanosine, andthe like, linked to a pentose at the 1′ position, including 2′-deoxy and2′-hydroxyl forms, e.g. as described in Kornberg and Baker, DNAReplication, 2nd Ed. (Freeman, San Francisco, 1992). The term“nucleotide” as used herein refers to a phosphate ester of a nucleoside,e.g., mono, di and triphosphate esters, wherein the most common site ofesterification is the hydroxyl group attached to the C-5 position of thepentose. “Analogs” in reference to nucleosides include syntheticnucleosides having modified base moieties and/or modified sugarmoieties, e.g. described generally by Scheit, Nucleotide Analogs (JohnWiley, New York, 1980). The terms “labeled nucleoside” and “labelednucleotide” refer to nucleosides and nucleotides which are covalentlyattached to an energy transfer dye through a linkage.

As used herein, the term “oligonucleotide” refers to linear polymers ofnatural or modified nucleoside monomers, including double and singlestranded deoxyribonucleosides, ribonucleosides, α-anomeric formsthereof, and the like. Usually the nucleoside monomers are linked byphosphodiester linkages, where as used herein, the term “phosphodiesterlinkage” refers to phosphodiester bonds or analogs thereof includingphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate,phosphoramidate, and the like, including associated counterions, e.g.,H, NH₄, Na, and the like if such counterions are present. Theoligonucleotides range in size form a few monomeric units, e.g. 8-40, toseveral thousands of monomeric units. Whenever an oligonucleotide isrepresented by a sequence of letters, such as “ATGCCTG,” it will beunderstood that the nucleotides are in 5′→3′ order from left to rightand that “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G”denotes deoxyguanosine, and “T” denotes thymidine, unless otherwisenoted.

Nucleoside labeling can be accomplished using any of a large number ofknown nucleoside labeling techniques using known linkages, linkinggroups, and associated complementary functionalities. The linkagelinking the dye and nucleoside should (i) be stable to oligonucleotidesynthesis conditions, (ii) not interfere with oligonucleotide-targethybridization, (iii) be compatible with relevant enzymes, e.g.,polymerases, ligases, and the like, and (iv) not quench the fluorescenceof the dye.

Preferably, the dyes are covalently linked to the 5-carbon of pyrimidinebases and to the 7-carbon of 7-deazapurine bases. Several suitable baselabeling procedures have been reported that can be used with theinvention, e.g. Gibson et al, Nucleic Acids Research, 15:6455-6467(1987); Gebeyehu et al, Nucleic Acids Research, 15:4513-4535 (1987);Haralambidis et al, Nucleic Acids Research, 15:4856-4876 (1987); Nelsonet al., Nucleosides and Nucleotides, 5(3):233-241 (1986); Bergstrom etal., JACS, 111:374-375 (1989); U.S. Pat. Nos. 4,855,225, 5,231,191, and5,449,767, each of which is incorporated herein by reference.

Preferably, the linkages are acetylenic amido or alkenic amido linkages,the linkage between the dye and the nucleotide base being formed byreacting an activated N-hydroxysuccinimide (NHS) ester of the dye withan alkynylamino-, alkynylethoxyamino- or alkenylamino-derivatized baseof a nucleotide. More preferably, the resulting linkage isproargyl-1-ethoxyamido(3-(amino)ethoxy-1-propynyl),3-(carboxy)amino-1-propynyl or 3-amino-1-propyn-1-yl.

Several preferred linkages for linking the dyes of the invention to anucleoside base are —C≡C—CH₂—NH—C(O)—, 3-amino-1-propyn-1-yl,—C≡C—CH₂—NH—C(O)—(CH₂)₅—NH—C(O)—, —CH═CH—C(O)—NH—(CH₂)₅—NH—C(O)— and—C≡C—CH₂—O—CH₂CH₂NR₁R₂, where R₁ and R₂ taken separately are H, alkyl, aprotecting group or a fluorescent dye.

The synthesis of alkynylamino-derivatized nucleosides is taught by Hobbset al. in European Patent Application No. 87305844.0, and Hobbs et al.,J. Org. Chem., 54:3420 (1989), which is incorporated herein byreference. Briefly, the alkynylamino-derivatized nucleotides are formedby placing the appropriate halodideoxynucleoside (usually5-iodopyrimidine and 7-iodo-7-deazapurine dideoxynucleosides as taughtby Hobbs et al. (cited above)) and Cu(I) in a flask, flushing with argonto remove air, adding dry DMF, followed by addition of an alkynylamine,triethyl-amine and Pd(0). The reaction mixture can be stirred forseveral hours, or until thin layer chromatography indicates consumptionof the halodideoxynucleoside. When an unprotected alkynylamine is used,the alkynylamino-nucleoside can be isolated by concentrating thereaction mixture and chromatographing on silica gel using an elutingsolvent which contains ammonium hydroxide to neutralize the hydrohalidegenerated in the coupling reaction. When a protected alkynylamine isused, methanol/methylene chloride can be added to the reaction mixture,followed by the bicarbonate form of a strongly basic anion exchangeresin. The slurry can then be stirred for about 45 minutes, filtered,and the resin rinsed with additional methanol/methylene chloride. Thecombined filtrates can be concentrated and purified byflash-chromatography on silica gel using a methanol-methylene chloridegradient. The triphosphates are obtained by standard techniques.

The synthesis of oligonucleotides labeled with an energy transfer dye ofthe present invention can be accomplished using any of a large number ofknown oligonucleotide labeling techniques using known linkages, linkinggroups, and associated complementary functionalities. For example,labeled oligonucleotides may be synthesized enzymatically, e.g., using aDNA polymerase or ligase, e.g., Stryer, Biochemistry, Chapter 24, W.H.Freeman and Company (1981), or by chemical synthesis, e.g., by aphosphoramidite method, a phosphite-triester method, and the like, e.g.,Gait, Oligonucleotide Synthesis, IRL Press (1990). Labels may beintroduced during enzymatic synthesis utilizing labeled nucleosidetriphosphate monomers, or introduced during chemical synthesis usinglabeled non-nucleotide or nucleotide phosphoramidites, or may beintroduced subsequent to synthesis.

Generally, if the labeled oligonucleotide is made using enzymaticsynthesis, the following procedure may be used. A template DNA isdenatured and an oligonucleotide primer is annealed to the template DNA.A mixture of deoxynucleoside triphosphates is added to the reactionincluding dGTP, dATP, dCTP, and dTTP where at least a fraction of one ofthe deoxynucleotides is labeled with a dye compound of the invention asdescribed above. Next, a polymerase enzyme is added under conditionswhere the polymerase enzyme is active. A labeled polynucleotide isformed by the incorporation of the labeled deoxynucleotides duringpolymerase strand synthesis. In an alternative enzymatic synthesismethod, two primers are used instead of one, one primer complementary tothe +strand and the other complementary to the-strand of the target, thepolymerase is a thermostable polymerase, and the reaction temperature iscycled between a denaturation temperature and an extension temperature,thereby exponentially synthesizing a labeled complement to the targetsequence by PCR, e.g., PCR Protocols, Innis et al. eds., Academic Press(1990).

Generally, if the labeled oligonucleotide is made using a chemicalsynthesis, it is preferred that a phosphoramidite method be used.Phosphoramidite compounds and the phosphoramidite method ofpolynucleotide synthesis are preferred in synthesizing oligonucleotidesbecause of the efficient and rapid coupling and the stability of thestarting materials. The synthesis is performed with the growingoligonucleotide chain attached to a solid support, so that excessreagents, which are in the liquid phase, can be easily removed byfiltration, thereby eliminating the need for purification steps betweencycles.

In view of the utility of phosphoramidite reagents in labelingnucleosides and oligonucleotides, the present invention also relates tophosphoramidite compounds which include an energy transfer dye of thepresent invention.

Detailed descriptions of the chemistry used to form oligonucleotides bythe phosphoramidite method are provided in Caruthers et al., U.S. Pat.No. 4,458,066; Caruthers et al., U.S. Pat. No. 4,415,732; Caruthers etal., Genetic Engineering, 4:1-17 (1982); Users Manual Model 392 and 394Polynucleotide Synthesizers, pages 6-1 through 6-22, Applied Biosystems,Part No. 901237 (1991), each of which are incorporated by reference intheir entirety.

The following briefly describes the steps of a typical oligonucleotidesynthesis cycle using the phosphoramidite method. First, a solid supportincluding a protected nucleotide monomer is treated with acid, e.g.,trichloroacetic acid, to remove a 5′-hydroxyl protecting group, freeingthe hydroxyl for a subsequent coupling reaction. An activatedintermediate is then formed by simultaneously adding a protectedphosphoramidite nucleoside monomer and a weak acid, e.g., tetrazole, tothe reaction. The weak acid protonates the nitrogen of thephosphoramidite forming a reactive intermediate. Nucleoside addition iscomplete within 30 s. Next, a capping step is performed which terminatesany polynucleotide chains that did not undergo nucleoside addition.Capping is preferably done with acetic anhydride and 1-methylimidazole.The internucleotide linkage is then converted from the phosphite to themore stable phosphotriester by oxidation using iodine as the preferredoxidizing agent and water as the oxygen donor. After oxidation, thehydroxyl protecting group is removed with a protic acid, e.g.,trichloroacetic acid or dichloroacetic acid, and the cycle is repeateduntil chain elongation is complete. After synthesis, the polynucleotidechain is cleaved from the support using a base, e.g., ammonium hydroxideor t-butyl amine. The cleavage reaction also removes any phosphateprotecting groups, e.g., cyanoethyl. Finally, the protecting groups onthe exocyclic amines of the bases and the hydroxyl protecting groups onthe dyes are removed by treating the polynucleotide solution in base atan elevated temperature, e.g., 55° C.

Any of the phosphoramidite nucleoside monomers may be dye-labeledphosphoramidites. If the 5′-terminal position of the nucleotide islabeled, a labeled non-nucleotide phosphoramidite of the invention maybe used during the final condensation step. If an internal position ofthe oligonucleotide is to be labeled, a labeled nucleotidicphosphoramidite of the invention may be used during any of thecondensation steps.

Subsequent to their synthesis, oligonucleotides may be labeled at anumber of positions including the 5′-terminus. See Oligonucleotides andAnalogs, Eckstein ed., Chapter 8, IRL Press (1991) and Orgel et al.,Nucleic Acids Research 11 (18):6513 (1983); U.S. Pat. No. 5,118,800,each of which are incorporated by reference.

Oligonucleotides may also be labeled on their phosphodiester backbone(Oligonucleotides and Analogs, Eckstein ed., Chapter 9) or at the3′-terminus (Nelson, Nucleic Acids Research 20(23):6253-6259, and U.S.Pat. Nos. 5,401,837 and 5,141,813, both patents hereby incorporated byreference. For a review of oligonucleotide labeling procedures see R.Haugland in Excited States of Biopolymers, Steiner ed., Plenum Press, NY(1983).

In one preferred post-synthesis chemical labeling method anoligonucleotide is labeled as follows. A dye including a carboxy linkinggroup is converted to the n-hydroxysuccinimide ester by reacting withapproximately 1 equivalent of 1,3-dicyclohexylcarbodiimide andapproximately 3 equivalents of N-hydroxysuccinimide in dry ethyl acetatefor 3 hours at room temperature. The reaction mixture is washed with 5%HCl, dried over magnesium sulfate, filtered, and concentrated to a solidwhich is resuspended in DMSO. The DMSO dye stock is then added in excess(10-20×) to an aminohexyl derivatized oligonucleotide in 0.25 Mbicarbonate/carbonate buffer at pH 9.4 and allowed to react for 6 hours,e.g., U.S. Pat. No. 4,757,141. The dye labeled oligonucleotide isseparated from unreacted dye by passage through a size-exclusionchromatography column eluting with buffer, e.g., 0.1 molar triethylamineacetate (TEAA). The fraction containing the crude labeledoligonucleotide is further purified by reverse phase HPLC employinggradient elution.

6.4 Methods Employing Dyes and Reagents of the Present Invention

The energy transfer dyes and reagents of the present invention may beused in a wide variety of methods for detecting the presence of acomponent in a sample by labeling the component in the sample with areagent containing the dye. In particular, the energy transfer dyes andreagents of the present invention are well suited for use in methodswhich combine separation and fluorescent detection techniques,particularly methods requiring the simultaneous detection of multiplespatially-overlapping analytes. For example, the dyes and reagents areparticularly well suited for identifying classes of oligonucleotidesthat have been subjected to a biochemical separation procedure, such aselectrophoresis, where a series of bands or spots of target substanceshaving similar physiochemical properties, e.g. size, conformation,charge, hydrophobicity, or the like, are present in a linear or planararrangement. As used herein, the term “bands” includes any spatialgrouping or aggregation of analytes on the basis of similar or identicalphysiochemical properties. Usually bands arise in the separation ofdye-oligonucleotide conjugates by electrophoresis.

Classes of oligonucleotides can arise in a variety of contexts. In apreferred category of methods referred to herein as “fragment analysis”or “genetic analysis” methods, labeled oligonucleotide fragments aregenerated through template-directed enzymatic synthesis using labeledprimers or nucleotides, e.g., by ligation or polymerase-directed primerextension; the fragments are subjected to a size-dependent separationprocess, e.g., electrophoresis or chromatography; and, the separatedfragments are detected subsequent to the separation, e.g., bylaser-induced fluorescence. In a particularly preferred embodiment,multiple classes of oligonucleotides are separated simultaneously andthe different classes are distinguished by spectrally resolvable labels.

One such fragment analysis method is amplified fragment lengthpolymorphisim detection (AmpFLP) and is based on amplified fragmentlength polymorphisms, i.e., restriction fragment length polymorphismsthat are amplified by PCR. These amplified fragments of varying sizeserve as linked markers for following mutant genes through families. Thecloser the amplified fragment is to the mutant gene on the chromosome,the higher the linkage correlation. Because genes for many geneticdisorders have not been identified, these linkage markers serve to helpevaluate disease risk or paternity. In the AmpFLPs technique, thepolynucleotides may be labeled by using a labeled oligonucleotide PCRprimer, or by utilizing labeled nucleotide triphosphates in the PCR.

Another fragment analysis method is nick translation. Nick translationinvolves a reaction to replace unlabeled nucleotide triphosphates in adouble-stranded DNA molecule with labeled ones. Free 3′-hydroxyl groupsare created within the unlabeled DNA by “nicks” caused bydeoxyribonuclease I (DNAase I) treatment. DNA polymerase I thencatalyzes the addition of a labeled nucleotide to the 3′-hydroxylterminus of the nick. At the same time, the 5′ to 3′-exonucleaseactivity of this enzyme eliminates the nucleotide unit from the5′-phosphoryl terminus of the nick. A new nucleotide with a free 3′-OHgroup is incorporated at the position of the original excisednucleotide, and the nick is shifted along by one nucleotide unit in the3′ direction. This 3′ shift will result in the sequential addition ofnew labeled nucleotides to the DNA with the removal of existingunlabeled nucleotides. The nick-translated polynucleotide is thenanalyzed using a separation process, e.g., electrophoresis.

Another exemplary fragment analysis method is based on variable numberof tandem repeats, or VNTRs. VNTRs are regions of double-stranded DNAthat contain adjacent multiple copies of a particular sequence, with thenumber of repeating units being variable. Examples of VNTR loci arepYNZ22, pMCT118, and Apo B. A subset of VNTR methods are those methodsbased on the detection of microsatellite repeats, or short tandemrepeats (STRs), i.e., tandem repeats of DNA characterized by a short(2-4 bases) repeated sequence. One of the most abundant interspersedrepetitive DNA families in humans is the (dC-dA)n-(dG-dT)n dinucleotiderepeat family (also called the (CA)n dinucleotide repeat family). Thereare thought to be as many as 50,000 to 100,000 (CA)n repeat regions inthe human genome, typically with 15-30 repeats per block. Many of theserepeat regions are polymorphic in length and can therefore serve asuseful genetic markers. Preferably, in VNTR or STR methods, label isintroduced into the polynucleotide fragments by using a dye-labeled PCRprimer.

Another exemplary fragment analysis method is DNA sequencing. Ingeneral, DNA sequencing involves an extension/termination reaction of anoligonucleotide primer. Included in the reaction mixture aredeoxynucleoside triphosphates (dNTPs) which are used to extend theprimer. Also included in the reaction mixture is at least onedideoxynucleoside triphosphate (ddNTP) which when incorporated onto theextended primer prevents the further extension of the primer. After theextension reaction has been terminated, the different terminationproducts that are formed are separated and analyzed in order todetermine the positioning of the different nucleosides.

Fluorescent DNA sequencing may generally be divided into two categories,“dye primer sequencing” and “dye terminator sequencing”. In dye primersequencing, a fluorescent dye is incorporated onto the primer beingextended. Four separate extension/termination reactions are then run inparallel, each extension reaction containing a differentdideoxynucleoside triphosphate (ddNTP) to terminate the extensionreaction. After termination, the reaction products are separated by gelelectrophoresis and analyzed. See, for example, Ansorge et al., NucleicAcids Res. 15:4593-4602 (1987).

In one variation of dye primer sequencing, different primers are used inthe four separate extension/termination reactions, each primercontaining a different spectrally resolvable dye. After termination, thereaction products from the four extension/termination reactions arepooled, electrophoretically separated, and detected in a single lane.See, for example, Smith et al., Nature 321:674-679 (1986). Thus, in thisvariation of dye primer sequencing, by using primers containing a set ofspectrally resolvable dyes, products from more than oneextension/termination reactions can be simultaneously detected.

In dye terminator sequencing, a fluorescent dye is attached to each ofthe dideoxynucleoside triphosphates. An extension/termination reactionis then conducted where a primer is extended using deoxynucleosidetriphosphates until the labeled dideoxynucleoside triphosphate isincorporated into the extended primer to prevent further extension ofthe primer. Once terminated, the reaction products for eachdideoxynucleoside triphosphate are separated and detected. In oneembodiment, separate extension/termination reactions are conducted foreach of the four dideoxynucleoside triphosphates. In another embodiment,a single extension/termination reaction is conducted which contains thefour dideoxynucleoside triphosphates, each labeled with a different,spectrally resolvable fluorescent dye.

Thus according to one aspect of the invention, a method is provided forconducting dye primer sequencing using one or more oligonucleotidereagents of the present invention. According to this method, a mixtureof extended labeled primers are formed by hybridizing a nucleic acidsequence with a fluorescently labeled oligonucleotide primer in thepresence of deoxynucleoside triphosphates, at least onedideoxynucleoside triphosphate and a DNA polymerase. The fluorescentlylabeled oligonucleotide primer includes an oligonucleotide sequencecomplementary to a portion of the nucleic acid sequence being sequenced,and an energy transfer fluorescent dye attached to the oligonucleotide.

According to the method, the DNA polymerase extends the primer with thedeoxynucleoside triphosphates until a dideoxynucleoside triphosphate isincorporated which terminates extension of the primer. Aftertermination, the mixture of extended primers are separated. The sequenceof the nucleic acid sequence is then determined by fluorescentlydetecting the mixture of extended primers formed.

In a further embodiment of this method, four dye primer sequencingreactions are run, each primer sequencing reaction including a differentfluorescently labeled oligonucleotide primer and a differentdideoxynucleoside triphosphate (ddATP, ddCTP, ddGTP and ddTTP). Afterthe four dye primer sequencing reactions are run, the resulting mixturesof extended primers may be pooled. The mixture of extended primers maythen be separated, for example by electrophoresis and the fluorescentsignal from each of the four different fluorescently labeledoligonucleotide primers detected in order to determine the sequence ofthe nucleic acid sequence.

According to a further aspect of the invention, a method is provided forconducting dye terminator sequencing using one or more dideoxynucleosidetriphosphates labeled with an energy transfer dye of the presentinvention. According to this method, a mixture of extended primers areformed by hybridizing a nucleic acid sequence with an oligonucleotideprimer in the presence of deoxynucleoside triphosphates, at least onefluorescently labeled dideoxynucleotide triphosphate and a DNApolymerase. The fluorescently labeled dideoxynucleotide triphosphateincludes a dideoxynucleoside triphosphate labeled with an energytransfer fluorescent dye of the present invention.

According to this method, the DNA polymerase extends the primer with thedeoxynucleoside triphosphates until a fluorescently labeleddideoxynucleoside triphosphate is incorporated into the extended primer.After termination, the mixture of extended primers are separated. Thesequence of the nucleic acid sequence is then determined by detectingthe fluorescently labeled dideoxynucleoside attached to the extendedprimer.

In a further embodiment of this method, the step of forming a mixture ofextended primers includes hybridizing the nucleic acid sequence withfour different fluorescently labeled dideoxynucleoside triphosphates,i.e., a fluorescently labeled dideoxycytosine triphosphate, afluorescently labeled dideoxyadenosine triphosphate, a fluorescentlylabeled dideoxyguanosine triphosphate, and a fluorescently labeleddideoxythymidine triphosphate.

In each of the above-described fragment analysis methods, the labeledoligonucleotides are preferably separated by electrophoretic procedures,e.g. Gould and Matthews, cited above; Rickwood and Hames, Eds., GelElectrophoresis of Nucleic Acids: A Practical Approach, (IRL PressLimited, London, 1981); or Osterman, Methods of Protein and Nucleic AcidResearch, Vol. 1 Springer-Verlag, Berlin, 1984). Preferably the type ofelectrophoretic matrix is crosslinked or uncrosslinked polyacrylamidehaving a concentration (weight to volume) of between about 2-20 weightpercent. More preferably, the polyacrylamide concentration is betweenabout 4-8 percent. Preferably in the context of DNA sequencing inparticular, the electrophoresis matrix includes a strand separating, ordenaturing, agent, e.g., urea, formamide, and the like. Detailedprocedures for constructing such matrices are given by Maniatis et al.,“Fractionation of Low Molecular Weight DNA and RNA in PolyacrylamideGels Containing 98% Formamide or 7 M Urea,” in Methods in Enzymology,65:299-305 (1980); Maniatis et al., “Chain Length Determination of SmallDouble- and Single-Stranded DNA Molecules by Polyacrylamide GelElectrophoresis,” Biochemistry, 14:3787-3794 (1975); Maniatis et al.,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory,New York, 1982), pgs. 179-185; and ABI PRISM™ 377 DNA Sequencer User'sManual, Rev. A, January 1995, Chapter 2 (p/n 903433, The Perkin-ElmerCorporation, Foster City, Calif.), each of which are incorporated byreference. The optimal polymer concentration, pH, temperature,concentration of denaturing agent, etc. employed in a particularseparation depends on many factors, including the size range of thenucleic acids to be separated, their base compositions, whether they aresingle stranded or double stranded, and the nature of the classes forwhich information is sought by electrophoresis. Accordingly applicationof the invention may require standard preliminary testing to optimizeconditions for particular separations. By way of example,oligonucleotides having sizes in the range of between about 20-300 baseshave been separated and detected in accordance with the invention in thefollowing matrix: 6 percent polyacrylamide made from 19 parts to 1 partacrylamide to bis-acrylamide, formed in a Tris-borate EDTA buffer at pH8.3.

After electrophoretic separation, the dye-oligonucleotide conjugates aredetected by measuring the fluorescence emission from the dye labeledpolynucleotides. To perform such detection, the labeled polynucleotidesare illuminated by standard means, e.g. high intensity mercury vaporlamps, lasers, or the like. Preferably the illumination means is a laserhaving an illumination beam at a wavelength between 488 and 550 nm. Morepreferably, the dye-polynucleotides are illuminated by laser lightgenerated by an argon ion laser, particularly the 488 and 514 nmemission lines of an argon ion laser, or an the 532 emission line of aneodymium solid-state YAG laser. Several argon ion lasers are availablecommercially which lase simultaneously at these lines, e.g. Cyonics,Ltd. (Sunnyvale, Calif.) Model 2001, or the like. The fluorescence isthen detected by a light-sensitive detector, e.g., a photomultipliertube, a charged coupled device, or the like.

6.5 Kits Incorporating the Energy Transfer Dyes

The present invention also relates to kits having combinations of energytransfer fluorescent dyes and/or reagents. In one embodiment, the kitincludes at least two spectrally resolvable energy transfer dyesaccording to the present invention. In this kit, the energy transferdyes preferably include the same donor dye so that a single light sourceis needed to excite the dyes.

In another embodiment, the kit includes dideoxycytosine triphosphate,dideoxyadenosine triphosphate, dideoxyguanosine triphosphate, anddideoxythymidine triphosphate, each dideoxynucleotide triphosphatelabeled with an energy transfer dye according to the present invention.In one embodiment, each energy transfer dye is spectrally resolvablefrom the other energy transfer dyes attached to the otherdideoxynucleotide triphosphates. In this kit, the energy transfer dyespreferably include the same first xanthene dye.

In yet another embodiment, the kit includes at least twooligonucleotides, each oligonucleotide including an energy transfer dyeaccording to the present invention. In one embodiment, eacholigonucleotide contains an energy transfer dye which is spectrallyresolvable from the energy transfer dyes attached to the otheroligonucleotides. In another embodiment, the kit includes at least fouroligonucleotides which each contain a spectrally resolvable energytransfer dye.

7. EXAMPLES

The energy transfer fluorescent dyes and their use in DNA sequencing isillustrated by the following examples. Further objectives and advantagesother than those set forth above will become apparent from the examples.

Example 1 Synthesis of 5TMR-B-CF

5TMR-B-CF was synthesized from 5-TMR NHS and4′-aminomethyl-5-carboxyfluorescein according to the reaction sequencesdescribed in Examples 1A-C. 5TMR-B-CF was then converted to5TMR-B-CF-NHS according to the reaction sequence described in 1D so thatthe dye could be coupled to a nucleoside, nucleotide or oligonucleotideprimer.

Example 1A Synthesis of 5-TMR-B

A mixture of 4-aminomethylbenzoic acid (3 mg, 19 μmol), 5-TMR NHS (5 mg,9 μmol) and triethylamine (20 μL) was suspended in dimethylformamide(DMF, 200 μL) in a 1.5-mL eppendorf tube. The mixture was heated to 60°C. for 10 minutes. Reaction progress was monitored by thin layerchromatography (TLC) on silica gel with elution with a 400/30/10 mixtureof dichloromethane, methanol and acetic acid. The insoluble4-aminomethylbenzoic acid was separated by centrifugation and the DMFsolution was decanted into 5% HCl (1 mL). The insoluble 5TMR-B wasseparated by centrifugation, washed with 5% HCl (2×1 mL) and dried in avacuum centrifuge. The product was dissolved in DMF (200 μL) and used toprepare 5TMR-B-NHS.

Example 1B Synthesis of 5-TMR-B-NHS

A solution of 5TMR-B in DMF (125 μL), diisopropylethylamine (10 μL) anddisuccinimidylcarbonate (10 mg) was combined in a 1.5-mL eppendorf tubeand heated to 60° C. The reaction progress was monitored by TLC onsilica gel with elution with a 600/60/16 mixture of dichloromethane,methanol and acetic acid. After five minutes, the reaction appeared tobe complete. The solution was diluted into methylene chloride (3 mL) andwashed with 250 mM carbonate/bicarbonate buffer (pH 9, 4×1 mL), dried(Na₂SO₄) and concentrated to dryness on a vacuum centrifuge. The solidwas dissolved in DMF (100 μL). The yield was determined by diluting analiquot into pH 9 buffer and measuring the absorbance at 552 nm. Usingan extinction coefficient of 50,000 cm⁻¹ M⁻¹, the concentration of5TMR-B-NHS was 4.8 mM. Yield from 5TMR NHS was 8%.

Example 1C Synthesis of 5TMR-B-CF

A solution of 5TMR-B-NHS (1 μmol in 250 μL DMF) was combined with asolution of 4′-aminomethyl-5-carboxyfluorescein (CF, 2.2 μmol in 100 μLDMSO) and triethylamine (20 μL) in a 1.5-mL eppendorf tube. The reactionwas monitored by HPLC using a C8 reverse-phase column with a gradientelution of 15% to 35% acetonitrile vs. 0.1 M triethylammonium acetate.HPLC analysis indicated the 5TMR-B-NHS was consumed, leaving the excess,unreacted CF. The reaction was diluted with 5% HCl (1 mL) and theproduct separated by centrifugation, leaving the unreacted CF in theaqueous phase. The solid was washed with 5% HCl (4×1 mL), dried in avacuum centrifuge and taken up in DMF (300 μL). The yield wasquantitative.

Example 1D Synthesis of 5-TMR-B-CF-NHS

A solution of 5TMR-B-CF (0.6 μmol in 100 μL DMF),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (DEC, 2 mg)and N-hydroxysuccinimide (4 mg) were combined in a 1.5-mL eppendorftube. The mixture was sonicated briefly and heated to 60° C. Thereaction was monitored by TLC on silica gel with elution with a600/60/16 mixture of dichloromethane, methanol and acetic acid. Thereaction was complete in 30 minutes and diluted with 5% HCl. The productwas separated by centrifugation and dried in a vacuum centrifuge. Theactivated dye was dissolved in DMF (20 μL).

Example 2 Synthesis of 5ROX-CF

A solution of 5ROX NHS (2 μmol in 100 μL DMSO) was mixed with CF (2 μmolin 100 μL DMSO) and triethylamine (10 μL). The reaction was followed byHPLC on a C8 reverse phase column using a gradient elution of 20% to 40%acetonitrile vs. 0.1 M TEAA. The reaction was diluted into 5% HCl (1 mL)and the product collected by centrifugation, washed with 5% HCl (1×1 mL)and dried in a vacuum centrifuge. The product was taken up in DMF (200μL).

Example 3 Synthesis of Cy5-CF

A solution of CF (0.4 μmol in 20 μL CMSO) and triethylamine (2 μL) wasadded to monoCy5 NHS (approximately 0.3 μmol). The reaction was followedby HPLC on a C8 reverse phase column using a gradient elution of 10% to30% acetonitrile vs. 0.1 M TEAA. The reaction was diluted into 5% HCl (1mL) and the product collected by centrifugation, washed with 5% HCl (1×1mL) and dried in a vacuum centrifuge. The product was taken up in DMF(100 μL).

Example 4 Comparison Of Fluorescence Strength of Energy Transfer Dyes

The following example compares the fluorescence emission strength of aseries of energy transfer dyes according to the present invention. Dyesolutions of 5TMR, 6TMR-CF, 5TMR-gly-CF, 5TMR-CF, 5TMR-B-CF,5TMR-gly-5AMF, 5TMR-5AMF and 5TMR-lys-5FAM were measured in 1×TBE/8Murea. Each dye solution had an optical density of 0.1 at 560 nm and wasexcited at 488 nm.

TABLE 7

The structures of each of these dyes is illustrated in Table 7. FIG. 2provides a bar graph of the relative fluorescence of each of these dyes.

As can be seen from FIG. 2, energy transfer dyes where the linker isattached to the acceptor at the 5 ring position (5TMR-CF and 5-TMR-B-CFwere found to exhibit significantly stronger fluorescence than theacceptor dye itself or when the acceptor dye is linked at the 6 ringposition (6TMR-CF). As also can be seen from FIG. 2, energy transferdyes where the linker has the formula R₁XC(O)R₂ where R₂ is benzene(5TMR-B-CF) were found to have significantly enhanced fluorescence ascompared to the dye where the linker has the formula —CH₂NHCO— (5TMR-CF)or —CH₂NHCOCH₂NHCO— (5TMR-gly-5AMF).

As can also be seen from FIG. 2, energy transfer dyes where the linkeris attached to both the donor and acceptor at the 5 ring position(5TMR-5AMF and 5TMR-gly-5AMF) were found to have significantfluorescence. Interestingly, the use of a lysine linker was found not toresult in appreciable energy transfer between the donor and acceptor.

Example 5 Dye Primer Sequencing Using Energy Transfer Dye

In this example, dye primer sequencing was performed on M13 (SEQ. ID.NO.: 1) in order to compare the relative brightness of 5TMR-CF and5TMR-B-CF labeled oligonucelotides. In this example, dye primersequencing was performed according to the ABI PRISM™ 377 DNA SequencerUser's Manual, Rev. B, January 1995, Chapter 2 (p/n 402114, ThePerkin-Elmer Corporation, Foster City, Calif.). 5TMR-CF and 5TMR-B-CFwere each attached to the 5′ end of M13-21 primer (SEQ. ID. NO.:2).Equimolar solutions of each primer were mixed with the M13 (SEQ. ID.NO.: 1) and sequenced with a single dideoxy nucleotide mixture(ddA/dNTP) and Taq FS. A plot of the resulting mixture ofoligonucleotides that were detected using 5TMR-CF and 5TMR-B-CF labeledprimers is presented in FIG. 7. As can be seen from FIG. 7,oligonucleotides labeled with 5TMR-B-CF are brighter thanoligonucleotides labeled with 5TMR-CF. As can also be seen from FIG. 7,the mobility of oligonucleotides labeled with 5TMR-B-CF are about onenucleotide slower than the oligonucleotides labeled with 5TMR-CF.

Example 6 Dye Primer Sequencing Using Four Dyes

Dye primer sequencing was performed on the M13 (SEQ ID NO: 1) using aset of four dyes attached to the M13-21 primer (SEQ ID NO: 2) asdescribed in Example 5. FIG. 8 is a four color plot of the dye labeledoligonucleotides produced from the sequencing. The peak for cytosinecorresponds to the fluorescence of 5-carboxy-R110. The peak foradenosine corresponds to the fluorescence of 5-carboxy-R6G. The peak forguanosine corresponds to the fluorescence of TMR-B-CF. The peak forthymidine corresponds to the fluorescence of ROX-CF.

As can be seen from FIG. 8, each of the dye labeled oligonucleotidesexhibit significant fluorescence intensity. In addition, the differentdye labeled oligonucleotides exhibit sufficiently similar mobility sothat good resolution of the series of peaks is achieved.

Example 7 Synthesis of 6-CFB-DTMR-2-NHS

6-CFB-DTMR-2 was synthesized from DTMR-2 and 6-CFB according to thereaction sequences described in Examples 1A-B. 6-CFB-DTMR-2 was thenconverted to 6-CFB-DTMR-2-NHS according to the reaction sequencedescribed in 1C so that the dye could be coupled to a nucleoside,nucleotide or oligonucleotide primer.

Example 7A Synthesis of DTMR-2-NHS

A solution of DTMR-2 in DMF, N-hydroxysuccinimide and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride werecombined in an eppendorf tube and heated to 60° C. The reaction progresswas monitored by TLC on silica gel. After the reaction appeared to becomplete, the solution was diluted into methylene chloride and washedwith 250 mM carbonate/bicarbonate buffer (pH 9, 4×1 mL), and then withan HCl solution (5%, 1×1 mL), dried (Na₂SO₄) and concentrated to drynesson a vacuum centrifuge.

Example 7B Synthesis of 6-CF-B-DTMR-2

A solution of 6-CFB in dimethylsulfoxide (100 μL 11 mM) was combinedwith a solution of DTMR-2 succidimidyl ester in dimethylformamide (100μL 22 mM) and triethylamine (20 μL). The reaction was added to asolution of hydrochloric acid (5%, 1 mL) and the solid separated bycentrifugation. The red solid was dissolved in carbonate/bicarbonatebuffer (250 mM, pH 9, 100 μL) and reprecipitated with dilute HCl. Thesolid was dried in a vacuum centrifuge and dissolved indimethylformamide (200 μL). The concentration of the dye solution wasdetermined by diluting an aliquot into 40% acetonitrile/0.1 Mtriethylammonium acetate buffer (pH 7). Assuming an extinctioncoefficient of 80,000 cm⁻¹ m⁻¹ for fluorescein, the 6-CF-B-DTMR-2solution was found to be 4 mM (70% yield).

Example 7C Synthesis of 6-CF-B-DTMR-NHS

A solution of 6-CF-B-DTMR-2 in dimethylformamide (200 μL, 4 mM) wasadded N-hydroxysuccinimide (10 mg) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5 μg).Additional N-hydroxysuccinimide (10 mg) was added. The reaction progresswas monitored by thin-layer chromatography on silica gel with elutionwith dichloromethane: methanol:acetic acid in a 600:60:16 mixture. Whenthe reaction was complete, dilute HCl (5%, 1 mL) was added and theproduct separated by centrifugation. The solid was dried in a vacuumcentrifuge and dissolved in dimethylformamide (100 μL). Theconcentration of the dye solution was determined by diluting an aliquotinto 40% acetonitrile/0.1 M triethylammonium acetate buffer (pH 7).Assuming an extinction coefficient of 80,000 cm⁻¹m⁻¹ for fluorescein,the 6-CF-B-DTMR-NHS solution was found to be 5.4 mM (68% yield).

Example 8 Comparison Of Fluorescence Strength of Dyes

The following example compares the fluorescence emission strength of aseries of energy transfer dyes according to the present invention to thecorresponding acceptor dye. According to this example, each dye wasattached to a 21 primer sequence (5′-TGTAAAACGACGGCCAGT) (SEQ ID NO: 1)with an aminohexyl linkage at the 5′ end. The oligonucleotides werequantitated based on the absorbance at 260 nm, assuming an extinctioncoefficient of 180,000 cm⁻¹ M⁻¹. Spectra were obtained at a primerconcentration of 0.4 μM in 8M urea, 1× Tris/Borate/EDTA (TBE) bufferwith 488 nm excitation. FIG. 9A provides the overlaid spectra of5-CFB-DR110-2 and DR110-2. FIG. 9B provides the overlaid spectra of5-CFB-DR6G-2 and DR6G-2. FIG. 9C provides the overlaid spectra of6-CFB-DTMR-2 and DTMR-2. FIG. 9D provides the overlaid spectra of6-CFB-DROX-2 and DROX-2.

The structures of each of these dyes is illustrated in Table 1. As canbe seen from FIGS. 9A-D, energy transfer dyes were found to exhibitsignificantly stronger fluorescence than the acceptor dye itself.

FIG. 10 shows the normalized fluorescence emission spectra of fourdye-labeled oligonucleotides. Spectra were obtained at a primerconcentration of 0.4 μM in 8M urea, 1× Tris/Borate/EDTA (TBE) bufferwith 488 nm excitation. The dyes shown in FIG. 10 include5-CFB-DR110-2,5-CFB-DR6G-2,6-CFB-DTMR-2, and 6-CFB-DROX-2. As can beseen from FIG. 10, all four energy transfer dyes are well resolvedrelative to each other.

Example 9 Dye Primer Sequencing Using Energy Transfer Dye

In this example, dye primer sequencing was performed on M13 (SEQ ID NO:2) using 5-CF-TMR-2,5-CF-B-TMR-2,6-CF-B-DTMR-2 and DTMR-2 labeledprimers. In this example, dye primer sequencing was performed accordingto the ABI PRISM™ 377 DNA Sequencer User's Manual, Rev. B, January 1995,Chapter 2 (p/n 402114, The Perkin-Elmer Corporation, Foster City,Calif.). The dye was attached to the 5′ end of M13-21 primer (SEQ IDNO:3). Equimolar solutions of each primer were mixed with the M13 (SEQID NO: 2) and sequenced with a single dideoxy nucleotide mixture(ddA/dNTP) and Taq FS. Plots of the resulting mixtures ofoligonucleotides that were detected using 5-CF-TMR-2 and 5-CF-B-TMR-2labeled primers are presented in FIG. 11. As can be seen from thisfigure, 5-CF-B-TMR-2 provides a significantly stronger signal than5-CF-TMR-2, showing the fluorescence enhancement provided by the linkerused in 5-CF-B-TMR-2.

Plots of the resulting mixtures of oligonucleotides that were detectedusing 6-CF-B-DTMR-2 and DTMR-2 labeled primers are presented in FIG. 12.As can be seen from this figure, 6-CF-B-DTMR-2 provides a significantlystronger signal than DTMR-2, showing the fluorescence enhancementprovided by the energy transfer dye.

Example 10 Dye Primer Sequencing Using Four Dyes

Dye primer sequencing was performed on M13 (SEQ ID NO:2) using a set offour dyes attached to the M13-21 primer (SEQ ID NO:3) as described inExample 5. FIG. 13 is a four color plot of the dye labeledoligonucleotides produced from the sequencing. The peak for cytosinecorresponds to the fluorescence of 5-CFB-DR110-2. The peak for adenosinecorresponds to the fluorescence of 6-CFB-DR6g-2. The peak for guanosinecorresponds to the fluorescence of 5-CFB-DTMR-2. The peak for thymidinecorresponds to the fluorescence of 5-CFB-DROX-2.

As can be seen from FIG. 13, each of the dye labeled oligonucleotidesexhibit significant fluorescence intensity. In addition, the differentdye labeled oligonucleotides exhibit sufficiently similar mobility sothat good resolution of the series of peaks is achieved.

The foregoing description of preferred embodiments of the invention hasbeen presented for purposes of illustration and description. It is notintended to be exhaustive or to limit the invention to the precise formsdisclosed. Obviously, many modifications and variations will be apparentto practitioners skilled in this art and are intended to fall within thescope of the invention.

1. A fluorescently labeled reagent comprising an oligonucleotide oroligonuclotide analog and an energy transfer dye, wherein energytransfer dye is covalently linked to the oligonucleotide oroligonucleotide analog and comprises: a xanthene donor dye capable ofabsorbing light at a first wavelength and emitting excitation energy inresponse thereto; a 4,7-dichlororhodamine acceptor dye capable ofabsorbing the excitation energy emitted by the donor dye and fluorescingat a second wavelength in response thereto; and a non-nucleosidic linkerlinking the xanthene donor dye to the 4′-, 5- or 6-position of the4,7-dichlororhodamine acceptor dye.
 2. The fluorescently labeled reagentof claim 1 in which the linker has a backbone that is less than 9 atomsin length.
 3. The fluorescently labeled reagent of claim 1 in which thelinker comprises a functional group selected from an alkene, a diene, analkyne, a five membered ring having at least one unsaturated bond, a sixmembered ring having at least one unsaturated bond and a fused ringstructure.
 4. The fluorescently labeled reagent of claim 1 in which theenergy transfer dye is covalently linked to the 5′-terminus of theoligonucleotide or oligonucleotide analog.
 5. The fluorescently labeledreagent of claim 1 in which the energy transfer dye is covalently linkedto the 3′-terminus of the oligonucleotide or oligonucleotide analog. 6.The fluorescently labeled reagent of claim 1 in which the energytransfer dye is covalently linked to a base moiety of theoligonucleotide or oligonucleotide analog.
 7. The fluorescently labeledreagent of claim 1 in which the energy transfer dye is covalently linkedto the base moiety by way of an acetylenic amido or alkenoic amidolinkage.
 8. The fluorescently labeled reagent of claim 7 in which theacetylenic amido or alkenoic amido linkage is selected from—C≡C—CH₂—NH—C(O)—, 3-amino-1-propyn-1-yl, —C≡C—CH₂—NH—C(O)—(CH₂)₅—C(O)—,—C═CH—C(O)—NH—(CH₂)₅—NH—C(O)— and —C≡C—CH₂—O—CH₂—CH₂-NR—, where R ishydrogen, a protecting group or alkyl.
 9. The fluorescently labeledreagent of claim 1 in which the xanthene donor dye is a fluorescein dye.10. The fluorescently labeled reagent of claim 9 in which thenon-nucleosidic linker links the 4′-position of the fluorescein donordye to the 5- or 6-position of the 4,7-dichlororhodamine acceptor dye.11. The fluorescently labeled reagent of claim 10 in which energytransfer dye is covalently linked to the oligonucleotide oroligonucleotide analog via the 5- or 6-position of the fluorescein donordye or the 4′-position of the 4,7-dichlororhodamine acceptor dye. 12.The fluorescently labeled reagent of claim 10 in which the4,7-dichlororhodamine acceptor dye comprises structure (I):

wherein: R¹, R², R³ and R⁴ are each, independently of one another,selected from hydrogen and alkyl, or alternatively R¹ and R⁵, R² and R⁶,R³ and R⁸ and/or R⁴ and R⁹ are taken together with the atoms to whichthey are bonded to form a 5, 6 or 7-membered ring; R⁵, R⁶, R⁷, R⁸, R⁹and R¹⁰ are each, independently of one another, selected from hydrogen,fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne,sulfonate, sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl andsubstituted phenyl, or alternatively, R⁶ and R⁷ and/or R⁹ and R¹⁰ aretaken together with the atoms to which they are bonded to form a benzogroup; X¹, X³ and X⁴ are each, independently of one another, selectedfrom hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl,alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile andalkoxy; and one of X³ or X⁴ comprises the non-nucleosidic linker linkingthe fluorescein donor dye.
 13. The fluorescently labeled reagent ofclaim 10 in which the fluorescein donor dye comprises structure (II):

wherein: R¹¹, R¹², R¹³, R¹⁵ and R¹⁶ are each, independently of oneanother, selected from hydrogen, fluorine, chlorine, bromine, iodine,carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium,amido, nitrile, alkoxy, phenyl and substituted phenyl, or alternatively,R¹² and R¹³ and/or R¹⁵ and R¹⁶ are taken together with the atoms towhich they are bonded to form a benzo group; X¹, X¹², X¹³, X¹⁴ and X¹⁵are each, independently of one another, selected from hydrogen,fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne,sulfonate, sulfone, amino, ammonium, amido, nitrile and alkoxy; and R¹⁴comprises the non-nucleosidic linker linking the 4,7-dichlororhodamineacceptor dye.
 14. The fluorescently labeled reagent of claim 10 in whichenergy transfer dye comprises structure (IV):

wherein: R¹, R², R³ and R⁴ are each, independently of one another,selected from hydrogen, methyl and ethyl, or alternatively R¹ and R⁵, R²and R⁶, R³ and R⁵ and/or R⁴ and R⁹ are taken together with the atoms towhich they are bonded to form a 5, 6 or 7-membered ring; R⁵, R⁶, R⁷, R⁸,R⁹ and R¹⁰ are each, independently of one another, selected fromhydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene,alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, alkoxy,phenyl and substituted phenyl, or alternatively, R⁶ and R⁷ and/or R⁹ andR¹⁰ are taken together with the atoms to which they are bonded to form abenzo group; X¹ and X³ are each, independently of one another, selectedfrom hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl,alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile andalkoxy; L is the non-nucleosidic linker; R¹¹, R¹², R¹³, R¹⁵ and R¹⁶ areeach, independently of one another, selected from hydrogen, fluorine,chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate,sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl and substitutedphenyl, or alternatively, R¹² and R¹³ and/or R¹⁵ and R¹⁶ are takentogether with the atoms to which they are bonded to form a benzo group;and X¹¹, X¹², X¹³, X¹⁴ and X¹⁵ are each, independently of one another,selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl,alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido,nitrile and alkoxy, with the proviso that one of R⁸, X¹³ or X¹⁴comprises a linkage that covalently links the energy transfer dye to theoligonucleotide or oligonucleotide analog.
 15. The fluorescently labeledreagent of claim 14 in which X¹ is carboxylate.
 16. The fluorescentlylabeled reagent of claim 15 in which R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,R¹⁰ and X³ are each hydrogen.
 17. The fluorescently labeled reagent ofclaim 15 in which R¹, R³, R⁵, R⁷, R⁸, R¹⁰ and X³ are each hydrogen; R²and R⁴ are each ethyl; and R⁶ and R⁹ are each methyl.
 18. Thefluorescently labeled reagent of claim 15 in which R¹, R², R³ and R⁴ areeach methyl; and R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen. 19.The fluorescently labeled reagent of claim 15 in which R¹ and R⁵, R² andR⁶, R³ and R⁸ and R⁴ and R⁹ are taken together with the atoms to whichthey are bonded to form six-membered rings; and R⁷, R¹⁰ and X³ are eachhydrogen.
 20. The fluorescently labeled reagent of claim 15 in which R¹,R³, R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen; R² is ethyl; andR⁶ is methyl.
 21. The fluorescently labeled reagent of claim 15 in whichR¹, R⁵, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen; R³, R⁴ and R⁶ are eachmethyl; and R² is ethyl.
 22. The fluorescently labeled reagent of claim15 in which R⁵, R⁶, R⁷, R¹⁰ and X³ are each hydrogen; R¹ and R² are eachmethyl; and R³ and R⁸ and R⁴ and R⁹ are taken together with the atoms towhich they are bonded to form six-membered rings.
 23. The fluorescentlylabeled reagent of claim 15 in which R¹, R², R⁵, R⁶, R⁷, R¹⁰ and X³ areeach hydrogen; and R³ and R⁸ and R⁴ and R⁹ are taken together with theatoms to which they are bonded to form six-membered rings.
 24. Thefluorescently labeled reagent of claim 15 in which R¹, R⁵, R⁶, R⁷, R¹⁰and X³ are each hydrogen; R² is ethyl; and R³ and R⁸ and R⁴ and R⁹ aretaken together with the atoms to which they are bonded to formsix-membered rings.
 25. The fluorescently labeled reagent of claim 15 inwhich R¹, R², R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen; and R³and R⁴ are each methyl.